Aptamers to von Willebrand Factor and their use as thrombotic disease therapeutics

ABSTRACT

The invention relates generally to the field of nucleic acids and more particularly to aptamers capable of binding to von Willebrand Factor useful as therapeutics in and diagnostics of thrombotic diseases and/or other diseases or disorders in which von Willebrand Factor mediated platelet aggregation has been implicated. The invention further relates to materials and methods for the administration of aptamers capable of binding to von Willebrand Factor.

REFERENCE TO RELATED APPLICATIONS

This non-provisional patent application claims priority under 35 U.S.C.§ 119(e) to the following provisional applications: U.S. ProvisionalPatent Application Ser. No. 60/608,047, filed Sep. 7, 2004, U.S.Provisional Patent Application Ser. No. 60/661,950, filed Mar. 11, 2005,U.S. Provisional Patent Application Ser. No. 60/678,427, filed May 6,2005, and U.S. Provisional Patent Application Ser. No. 60/690,231, filedJun. 13, 2005; each of which is herein incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The invention relates generally to the field of nucleic acids and moreparticularly to aptamers capable of binding to von Willebrand Factoruseful as therapeutics in and diagnostics of thrombotic diseases and/orother diseases or disorders in which von Willebrand Factor mediatedplatelet aggregation is implicated. The invention further relates tomaterials and methods for the administration of aptamers capable ofbinding to von Willebrand Factor.

BACKGROUND OF THE INVENTION

Aptamers are nucleic acid molecules having specific binding affinity tomolecules through interactions other than classic Watson-Crick basepairing.

Aptamers, like peptides generated by phage display or monoclonalantibodies (“mAbs”), are capable of specifically binding to selectedtargets and modulating the target's activity or binding interactions,e.g., through binding aptamers may block their target's ability tofunction. Discovered by an in vitro selection process from pools ofrandom sequence oligonucleotides, aptamers have been generated for over130 proteins including growth factors, transcription factors, enzymes,immunoglobulins, and receptors. A typical aptamer is 10-15 kDa in size(20-45 nucleotides), binds its target with nanomolar to sub-nanomolaraffinity, and discriminates against closely related targets (e.g.,aptamers will typically not bind other proteins from the same genefamily). A series of structural studies have shown that aptamers arecapable of using the same types of binding interactions (e.g., hydrogenbonding, electrostatic complementarities, hydrophobic contacts, stericexclusion) that drive affinity and specificity in antibody-antigencomplexes.

Aptamers have a number of desirable characteristics for use astherapeutics and diagnostics including high specificity and affinity,biological efficacy, and excellent pharmacokinetic properties. Inaddition, they offer specific competitive advantages over antibodies andother protein biologics, for example:

1) Speed and control. Aptamers are produced by an entirely in vitroprocess, allowing for the rapid generation of initial leads, includingtherapeutic leads. In vitro selection allows the specificity andaffinity of the aptamer to be tightly controlled and allows thegeneration of leads, including leads against both toxic andnon-immunogenic targets.

2) Toxicity and Immunogenicity. Aptamers as a class have demonstratedtherapeutically acceptable toxicity and lack of immunogenicity. Whereasthe efficacy of many monoclonal antibodies can be severely limited byimmune response to antibodies themselves, it is extremely difficult toelicit antibodies to aptamers most likely because aptamers cannot bepresented by T-cells via the MHC and the immune response is generallytrained not to recognize nucleic acid fragments.

3) Administration. Whereas most currently approved antibody therapeuticsare administered by intravenous infusion (typically over 2-4 hours),aptamers can be administered by subcutaneous injection (aptamerbioavailability via subcutaneous administration is >80% in monkeystudies (Tucker et al., J. Chromatography B. 732: 203-212, 1999)). Withgood solubility (>150 mg/mL) and comparatively low molecular weight(aptamer: 10-50 kDa; antibody: 150 kDa), a weekly dose of aptamer may bedelivered by injection in a volume of less than 0.5 mL. In addition, thesmall size of aptamers allows them to penetrate into areas ofconformational constrictions that do not allow for antibodies orantibody fragments to penetrate, presenting yet another advantage ofaptamer-based therapeutics or prophylaxis.

4) Scalability and cost. Therapeutic aptamers are chemically synthesizedand consequently can be readily scaled as needed to meet productiondemand. Whereas difficulties in scaling production are currentlylimiting the availability of some biologics and the capital cost of alarge-scale protein production plant is enormous, a single large-scaleoligonucleotide synthesizer can produce upwards of 100 kg/year andrequires a relatively modest initial investment. The current cost ofgoods for aptamer synthesis at the kilogram scale is estimated at$500/g, comparable to that for highly optimized antibodies. Continuingimprovements in process development are expected to lower the cost ofgoods to <$100/g in five years.

5) Stability. Therapeutic aptamers are chemically robust. They areintrinsically adapted to regain activity following exposure to factorssuch as heat and denaturants and can be stored for extended periods (>1yr) at room temperature as lyophilized powders.

Thrombotic Disease

During normal, controlled hemostasis, platelets do not adhere to healthyvessels, rather platelets typically adhere to the subendothelium ofinjured vessels. Platelet adhesion triggers a series of plateletactivation processes which ultimately results in thrombus formation andcessation of bleeding. The von Willebrand Factor (“vWF”) is a mediatorof platelet adhesion at sites of vascular damage. vWF is a largemulti-subunit, multimeric soluble factor mainly produced by vascularendothelial cells. The von Willebrand Factor becomes immobilized on theblood vessel wall via interactions between von Willebrand Factor domainA3 and exposed collagen. Transient interactions of the platelet-receptorglycoprotein Ib (“hereinafter GPIb”) and the A1 domain of theimmobilized von Willebrand Factor facilitates the adhesion andactivation of platelets at sites of vascular injury. E. G. Huizinga etal., Science, 297, 1176 (2002). Accordingly, the von Willebrand factoris pro-thrombotic, playing an important role during hemostasis infacilitating thrombus formation at sites of vascular injury.

Conversely, the von Willebrand Factor, by the same mechanism, also playsa key role in pathological conditions, such as cardiovascular diseases,involving platelet aggregation and thrombosis formation. Althoughantithrombotic therapies are currently available there is still a largeunmet need for additional therapies. The American Heart Associationestimates that more than 60 million people in the United States alonehave one or more forms of cardiovascular disease, and that a highproportion of people with cardiovascular disease are at higher risk forarterial thrombosis. S. P. Jackson and S. M. Schoenwaelder, NatureReviews, 2, 1-12 (2003).

A significant problem with presently available therapies is thatimproving efficacy reduces safety. S. P. Jackson and S. M.Schoenwaelder, Nature Reviews, 2, 1-12 (2003). Accordingly, it would bebeneficial to treat or prevent thrombotic disease by preventing plateletaggregation in the vasculature while minimizing bleeding side effects.The present invention provides materials and methods to meet these andother needs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the in vitro aptamer selection(SELEX™) process from pools of random sequence oligonucleotides.

FIG. 2 is an illustration depicting various PEGylation strategiesrepresenting standard mono-PEGylation, multiple PEGylation, andoligomerization via PEGylation.

FIG. 3 is a table listing the amino acid sequences of the von WillebrandFactor domain A1 proteins used in the experiments of the invention.

FIG. 4 is a table listing the amino acid sequence of the full lengthhuman von Willebrand Factor protein used in the experiments of theinvention.

FIG. 5 is an illustration depicting the proposed secondary structure ofARC1029 (SEQ ID NO 214).

FIG. 6 is a graph of the dot blot binding curves for ARC1029 (SEQ ID NO214) to full length vWF and rabbit vWF domain A1. A black box in thistable indicates a deletion.

FIG. 7 is a graph of FACS data showing that ARC1029 (SEQ ID NO 214)inhibits binding of human vWF and rabbit vWF A1 domain to lyophilizedhuman platelets.

FIG. 8 is a graph of an aggregometer trace showing ARC1029 (SEQ ID NO214) inhibiting botrocetin induced platelet aggregation over time.

FIG. 9 is a graphical representation of ARC1029 (SEQ ID NO 214)inhibiting botrocetin induced platelet aggregation.

FIG. 10 is an illustration depicting the proposed secondary structurefor the SEQ ID NO 217 wherein C=fC, T=fU, N=any nucleotide but in pairedregions it assumes a Watson/Crick base pair, and X=1 to 4 nucleotides orNx-Nx-Nx-Nx can be replaced with a PEG spacer.

FIG. 11 is an illustration depicting the sequence alignment for threeaptamers of the vWF rRdY SELEX™ Family #1.

FIG. 12 is an illustration depicting the proposed secondary structurefor SEQ ID NO 218.

FIG. 13 is an illustration depicting the proposed secondary structurefor SEQ ID NO 219.

FIG. 14 is an illustration depicting the sequence alignment for 26aptamers of the vWF DNA SELEX™ 2 Family #1. The black line at the top ofthe alignment represents the proposed core nucleic acid binding sequencerequired to bind the von Willebrand Factor target.

FIG. 15A is an illustration depicting the proposed secondary structurefor SEQ ID NO 220. FIG. 15B is an illustration depicting the secondarystructure of ARC1172 (SEQ ID NO 222) and which residues are tolerant of2′-OMe substitution.

FIG. 16 is a table depicting the nucleic acid sequences including anymodifications of ARC1029 (SEQ ID NO 214), ARC1115 (SEQ ID NO 221),ARC1172 (SEQ ID NO 222), and ARC1194 (SEQ ID NO 223) to ARC1243 (SEQ IDNO 272).

FIG. 17 is a table depicting the nucleic acid sequences including anymodifications of ARC1172 (SEQ ID NO 222), ARC1338 (SEQ ID NO 273) toARC1348 (SEQ ID NO 283) and ARC1361 (SEQ ID NO 284) to ARC1381 (SEQ IDNO 304). A black block in this table indicates a deletion. An “s”preceding a nucleotide indicator (e.g. T or dA) indicates aphosphorothioate substitution in the phosphate backbone 5′ to theindicated nucleotide.

FIG. 18 is a table depicting the nucleic acid sequences including anymodifications of ARC1172 (SEQ ID NO 222), ARC1524 (SEQ ID NO 305) toARC1535 (SEQ ID NO 316), ARC1546 (SEQ ID NO 317) and ARC1759 (SEQ ID NO318). A black block in this table indicates a deletion.

FIG. 19 is an illustration of the secondary structures of ARC1368 (SEQID NO 291) and ARC1534 (SEQ ID NO 315).

FIG. 20 is a graph depicting the clotting time, in the PFA-100 assay,for human whole blood treated with ARC1368 (SEQ ID NO 291) or ARC1525(SEQ ID NO 306) as a function of aptamer concentration.

FIG. 21 is a graph depicting occlusion time, in a PFA-100 assay, ofhuman whole blood treated with Integrilin™, ReoPro™ or ARC1368 (SEQ IDNO 291), as a function of drug concentration.

FIG. 22 is a graph depicting percent inhibition, in BIPA, of human PRP,treated with Integrilin™, ReoPro™ or ARC1368 (SEQ ID NO 291), as afunction of drug concentration.

FIG. 23 is a graph depicting percent inhibition, in AIPA, of human PRP,treated with Integrilin™, ReoPro™ or ARC1368 (SEQ ID NO 291), as afunction of drug concentration.

FIG. 24 is a graph depicting percentage of full length ARC1172 (SEQ IDNO 222) or ARC1368 (SEQ ID NO 291), detected in human plasma, as afunction of time.

FIG. 25 is a graph depicting primate plasma aptamer concentration(determined using Oligreen analysis) plotted as a function of timefollowing administration of ARC1368 (SEQ ID NO 291), ARC1779 (SEQ ID NO320) or ARC1780 (SEQ ID NO 321).

FIG. 26 is a graph showing the time points on the horizontal axis atwhich blood for testing was drawn from three cynomolgus macaques,ARC1779 (SEQ ID NO 320) plasma concentration (in nM) along the top thirdof the vertical axis, PFA-100 closure time (in seconds) on the middlethird of the vertical axis, and the template or cutaneous bleeding time(in minutes) on the bottom third of the vertical axis. The average fromall three animals for plasma aptamer concentration, PFA-100 closure timeand cutaneous bleeding time is plotted on the top third, middle thirdand bottom third of the graph, respectively.

FIG. 27 is a table showing the cutaneous bleeding time (CBT) in minutes,raw BIPA data and PFA-100 closure time (sec) at various time points,shown in column 1, relative to ARC1779 (SEQ ID NO 320) dosing in threedifferent cynomolgus macaques.

FIG. 28 is a graph showing the average PFA-100 closure time at varioustime points following ARC1779 (SEQ ID NO 320) dosing of C. macaques.

FIG. 29 is a graph showing the average bleeding time of the threeARC1779 (SEQ ID NO 320) treated macaques taken at various time pointsfollowing dosing.

FIG. 30 is a graph correlating the average bleeding time in ARC1779 (SEQID NO 320) treated C. macaques (left vertical axis) to the PFA-100closure time.

FIG. 31 is a schematic depicting the blood sample collection scheduleused in the assessment of ARC1779 (SEQ ID NO 320) in the cynomolgusmonkey electrolytic thrombosis model.

FIG. 32 is graph of ARC1779 (SEQ ID NO 320) plasma concentration(vertical axis) as a function of time in each cynomolgus monkey oftreatment group 3 tested in the electrolytic thrombosis model.

FIG. 33 is a graph of the time to occlusion of the right (hatched bar)or left carotid artery (indicated by a solid bar) in each cynomolgusmacaque from each treatment group tested in the cynomolgus monkeyelectrolytic thrombosis model. Bar pairs 1, 8 and 9 indicate treatmentgroup 1 (vehicle only) Bar pairs 2, 10, 11, 12 and 13 indicatestreatment groups 2 and 4 (ReoPro). Bar pairs 3 to 7 indicate treatmentgroup 3 (1000 nM aptamer plasma concentration target group). Bar pairs20, 22, 16, and 18 indicate treatment group 7 (750 nM plasma aptamerconcentration target group). Bar pairs 19, 21, 23 and 24 indicatetreatment group 6 (500 nM plasma aptamer concentration target group).Bar pairs 15 and 17 indicate treatment group 5 (300 nM plasma aptamerconcentration target group)

FIG. 34 is a graph showing the cutaneous bleed time in minutes (verticalaxis) of the various cynomolgous treatment groups in the electricalinjury model taken at the time points shown on the horizontal axis.

SUMMARY OF THE INVENTION

The present invention provides materials and methods for the treatmentof thrombotic disorders involving von Willebrand Factor mediatedplatelet aggregation.

The present invention provides aptamers that specifically bind to a vonWillebrand Factor target. In some embodiments, the von Willebrand Factortarget is human von Willebrand Factor. In some embodiments, the vonWillebrand Factor target is a variant of human von Willebrand Factorthat performs a biological function that is essentially the same as afunction of human von Willebrand Factor. In some embodiments, thebiological function of the von Willebrand Factor target or variantthereof is to mediate platelet aggregation. In some embodiments, thevariant of the human von Willebrand Factor target has substantially thesame structure and substantially the same ability to bind an aptamer ofthe invention as that of human von Willebrand Factor. In someembodiments, the vWF target is a non-human von Willebrand Factor. Insome embodiments, the aptamer of the invention binds the von WillebrandFactor target or a variant thereof that comprises an amino acid sequencewhich is at least 75%, 80%, 90% or 95% identical to SEQ ID NO 7 (FIG.4). In one embodiment, the von Willebrand Factor target comprises theamino acid sequence of SEQ ID NO 7.

The terms “sequence identity” or “% identity” in the context of two ormore nucleic acid or protein sequences, refer to two or more sequencesor subsequences that are the same or have a specified percentage ofamino acid residues or nucleotides that are the same, when compared andaligned for maximum correspondence, as measured using one of thefollowing sequence comparison algorithms or by visual inspection. Forsequence comparison, typically one sequence acts as a reference sequenceto which test sequences are compared. When using a sequence comparisonalgorithm, test and reference sequences are input into a computer,subsequence coordinates are designated if necessary, and sequencealgorithm program parameters are designated. The sequence comparisonalgorithm then calculates the percent sequence identity for the testsequence(s) relative to the reference sequence, based on the designatedprogram parameters. Optimal alignment of sequences for comparison can beconducted, e.g., by the local homology algorithm of Smith & Waterman,Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm ofNeedleman & Wunsch, J Mol. Biol. 48: 443 (1970), by the search forsimilarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visualinspection (see generally, Ausubel, F. M. et al., Current Protocols inMolecular Biology, pub. by Greene Publishing Assoc. andWiley-Interscience (1987).

One example of an algorithm that is suitable for determining percentsequence identity is the algorithm used in the basic local alignmentsearch tool (hereinafter “BLAST”), see, e.g. Altschul et al., J Mol.Biol. 215: 403-410 (1990) and Altschul et al., Nucleic Acids Res., 15:3389-3402 (1997). Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(hereinafter “NCBI”). The default parameters used in determiningsequence identity using the software available from NCBI, e.g., BLASTN(for nucleotide sequences) and BLASTP (for amino acid sequences) aredescribed in McGinnis et al., Nucleic Acids Res., 32: W20-W25 (2004). Ina preferred embodiment, percent identity is determined using the BLASTimplemented algorithm of Altschul et al., and the default parameters ofMcGinnis et al., which herein may be referred to as BLAST percentidentity.

In one embodiment, the vWF aptamer of the invention comprises adissociation constant for human von Willebrand Factor or a variantthereof, of about 100 nM or less, preferably 50 nM or less, preferably10 nM or less, preferably 5 nM or less, preferably 1 nM or less, andmore preferably 500 pM or less. The dissociation constant may bedetermined by dot blot assay using a multi-point titration and fittingthe equation y=(max/(1+K/protein))+yint as described in Example 1,below.

The present invention also provides an aptamer that specifically bindsto a von Willebrand Factor domain A1 target. In some embodiments, thevon Willebrand Factor domain A1 target is a human von Willebrand Factordomain A1 target. In some embodiments, the human von Willebrand Factordomain A1 target is a variant of human von Willebrand Factor domain A1that performs a biological function that is essentially the same as afunction of human von Willebrand Factor domain A1. In some embodiments,the biological function of von Willebrand Factor domain A1 or a variantthereof is to bind to platelets. In some embodiments, the variant ofhuman von Willebrand Factor domain target has substantially the samestructure and substantially the same ability to bind said aptamer asthat of human von Willebrand Factor domain A1. In other embodiments, thevon Willebrand Factor domain A1 target is non-human von WillebrandFactor domain A1 target, e.g. a rabbit or non-human primate vonWillebrand Factor domain A1 target.

In some embodiments, the von Willebrand Factor domain A1 target of theinvention comprises an amino acid sequence which is at least 75%, 80%,90% or 95% identical to any one of the sequences selected from the groupconsisting of: SEQ ID NOS 4 to 6. In a preferred embodiment, the vonWillebrand Factor domain A1 target comprises any one of the amino acidsequences selected from the group consisting: of SEQ ID NOS 4 to 6.

In one embodiment, the vWF aptamer of the invention comprises adissociation constant for human von Willebrand Factor domain A1 or avariant thereof, of about 100 nM or less, preferably 50 nM or less,preferably 10 nM or less, preferably 5 nM or less, preferably 1 nM orless, and more preferably 500 pM or less. In another embodiment, theaptamer of the invention comprises a dissociation constant for non-humanvon Willebrand Factor domain A1 or a variant thereof, of about 100 nM orless, preferably 50 nM or less, preferably 10 nM or less, preferably 5nM or less, preferably 1 nM or less, and more preferably 500 pM or less.The dissociation constant may be determined by dot blot assay using amulti-point titration and fitting the equationy=(max/(1+K/protein))+yint as described in Example 1, below.

In some embodiments, the invention provides an aptamer that specificallybinds to a von Willebrand Factor full length target. In someembodiments, the invention provides an aptamer that specifically bindsto a von Willebrand Factor full length target and a von WillebrandFactor domain A1 target. In some embodiments, the von Willebrand Factorfull length target is a human von Willebrand target or variant thereof.In other embodiments, the von Willebrand Factor full length target is anon-human von Willebrand target or variant thereof. In some embodiments,the von Willebrand Factor domain A1 target is a non-human von WillebrandFactor domain A1 target or variant thereof. In other embodiments, thevon Willebrand Factor domain A1 target is a human von Willebrand Factordomain A1 target or variant thereof. In some embodiments, the vonWillebrand Factor full length target or domain A1 target is selectedfrom the group consisting of: a rabbit, guinea pig, monkey, dog, sheep,mouse and rat, von Willebrand Factor full length or domain A1 target. Insome embodiments, the von Willebrand Factor full length target and vonWillebrand Factor domain A1 target to which the aptamer of the inventionspecifically binds, are from different species.

The present invention provides aptamers against a von Willebrand Factortarget that are ribonucleic acid or deoxyribonucleic acid or mixedribonucleic acid and deoxyribonucleic acid. Aptamers of the inventionmay be single stranded ribonucleic acid or deoxyribonucleic acid ormixed ribonucleic acid and deoxyribonucleic acid. In some embodiments,the aptamer of the invention comprises at least one chemicalmodification. In some embodiments, the chemical modification is selectedfrom the group consisting of: a chemical substitution at a sugarposition; a chemical substitution at a phosphate position; and achemical substitution at a base position, of the nucleic acid. In otherembodiments, the chemical modification is selected from the groupconsisting of: incorporation of a modified nucleotide, 3′ capping,conjugation to a high molecular weight, non-immunogenic compound,conjugation to a lipophilic compound, and incorporation ofphosphorothioate into the phosphate back bone. In a preferredembodiment, the non-immunogenic, high molecular weight compound ispolyalkylene glycol, more preferably polyethylene glycol.

In some embodiments of the present invention, an aptamer, e.g. a vonWillebrand Factor aptamer, that binds specifically to a target whereinthe aptamer comprises a nucleotide sequence having no more than four, nomore than three, no more than two or no more than one phosphorothioatebackbone modifications, and the aptamer has a binding affinity for thetarget wherein the binding affinity is increased relative to a secondaptamer having the same nucleotide sequence but lacking phosphorothioateback bone modification is provided. In some embodiments, the target is aprotein or peptide not having the known function of binding nucleicacid, particularly not have the known primary function of bindingnucleic acid.

In some embodiments, the aptamer of the invention modulates a functionof any one of the group consisting of: the von Willebrand Factor target,the von Willebrand Factor domain A1 target and a variant of eithertarget. In some embodiments, the modulated function is plateletaggregation mediation. In some embodiments, the aptamer of the inventioninhibits von Willebrand Factor mediated platelet aggregation in vivo. Inother embodiments, the aptamer of the invention prevents binding of anyone of the group consisting of: the von Willebrand Factor target, thevon Willebrand Factor domain A1 target and a variant thereof, to aplatelet. In other embodiments, the aptamer of the invention preventsbinding of any one of the group consisting of: the von Willebrand Factortarget, the von Willebrand Factor domain A1 target and a variant ofeither target, to a platelet receptor protein. In yet other embodiments,the aptamer of the invention prevents binding of any one of the groupconsisting of: the von Willebrand Factor target, the von WillebrandFactor domain A1 target and a variant of either target, to the plateletreceptor protein GPIb. In some embodiments, the aptamer of the inventionprevents vWF Factor mediated platelet aggregation while notsignificantly increasing bleeding time. In some embodiments, anon-significant increase in bleeding time is less than 15, minutes,preferably less than 10 minutes, more preferably less than 5 minutes,and in some embodiments, less than 3 minutes relative to the bleedingtime of a subject not treated with the aptamer of the invention. In someembodiments the bleeding time is determined by cutaneous (or template)bleeding time.

In some embodiments, the aptamer of the invention has substantially thesame ability to bind any one of the group consisting of: the vonWillebrand Factor target, the von Willebrand Factor domain A1 target anda variant thereof, as that of an aptamer selected from the groupconsisting of: SEQ ID NOS 11 to 50, SEQ ID NOS 54 to 94, SEQ ID NOS 98to 164, SEQ ID NO 165, SEQ ID NO 169, SEQ ID NO 172, SEQ ID NO 174, SEQID NO 177, SEQ ID NO 180, SEQ ID NO 183, SEQ ID NO 186, SEQ ID NO 189,SEQ ID NO 192, SEQ ID NO 198, SEQ ID NO 201, SEQ ID NO 205, SEQ ID NO208, SEQ ID NOS 212-214, ARC115 (SEQ ID NO 221), ARC1172 (SEQ ID NO222), ARC1194 (SEQ ID NO 223) to ARC1240 (SEQ ID NO 269), ARC1338(SEQ IDNO 273) to ARC1346(SEQ ID NO 281), ARC1361(SEQ ID NO 284) to ARC1381(SEQ ID NO 304), ARC1524 (SEQ ID NO 305), ARC1526 (SEQ ID NO 307) toARC1535 (SEQ ID NO 316), ARC1546 (SEQ ID NO 317), ARC1635 (SEQ ID NO319), ARC1759 (SEQ ID NO 318), ARC1779 (SEQ ID NO 320) to ARC1780 (SEQID NO 321) and ARC1884 (SEQ ID NO 322) to ARC1885 (SEQ ID NO 323). Inother embodiments, the aptamer of the invention has substantially thesame structure and substantially the same ability to bind of any one ofthe group consisting of: the von Willebrand Factor target, the vonWillebrand Factor domain A1 target and a variant thereof, as that of anaptamer selected from the group of sequences consisting of: SEQ ID NOS11 to 50, SEQ ID NOS 54 to 94, SEQ ID NOS 98 to 164, SEQ ID NO 165, SEQID NO 169, SEQ ID NO 172, SEQ ID NO 174, SEQ ID NO 177, SEQ ID NO 180,SEQ ID NO 183, SEQ ID NO 186, SEQ ID NO 189, SEQ ID NO 192, SEQ ID NO198, SEQ ID NO 201, SEQ ID NO 205, SEQ ID NO 208, SEQ ID NOS 212-214,ARC1115 (SEQ ID NO 221), ARC1172 (SEQ ID NO 222), ARC1194 (SEQ ID NO223) to ARC1240 (SEQ ID NO 269), ARC1338 (SEQ ID NO 273) to ARC1346 (SEQID NO 281), ARC1361 (SEQ ID NO 284) to ARC1381 (SEQ ID NO 304), ARC1524(SEQ ID NO 305), ARC1526 (SEQ ID NO 307) to ARC1535 (SEQ ID NO 316),ARC1546 (SEQ ID NO 317), ARC1635 (SEQ ID NO 319), ARC1759 (SEQ ID NO318), ARC1779 (SEQ ID NO 320) to ARC1780 (SEQ ID NO 321) and ARC1884(SEQID NO 322) to ARC1885(SEQ ID NO 323). In yet other embodiments, theaptamer of the invention is selected from the group consisting of: SEQID NOS 11 to 50, SEQ ID NOS 54 to 94, SEQ ID NOS 98 to 164, SEQ ID NO165, SEQ ID NO 169, SEQ ID NO 172, SEQ ID NO 174, SEQ ID NO 177, SEQ IDNO 180, SEQ ID NO 183, SEQ ID NO 186, SEQ ID NO 189, SEQ ID NO 192, SEQID NO 198, SEQ ID NO 201, SEQ ID NO 205, SEQ ID NO 208, SEQ ID NOS212-214, ARC1115 (SEQ ID NO 221), ARC1172 (SEQ ID NO 222), ARC1194 (SEQID NO 223) to ARC1240 (SEQ ID NO 269), ARC1338 (SEQ ID NO 273) toARC1346 (SEQ ID NO 281), ARC1361 (SEQ ID NO 284) to ARC1381 (SEQ ID NO304), ARC1524 (SEQ ID NO 305), ARC1526 (SEQ ID NO 307) to ARC1535 (SEQID NO 316), ARC1546 (SEQ ID NO 317), ARC1635 (SEQ ID NO 319), ARC1759(SEQ ID NO 318), ARC1779 (SEQ ID NO 320) to ARC1780 (SEQ ID NO 321) andARC1884 (SEQ ID NO 322) to ARC1885 (SEQ ID NO 323).

In a particular embodiment, the aptamer of the invention comprises theprimary nucleic acid sequence of ARC1172 (SEQ ID NO 222) or ARC1115 (SEQID NO 221) or ARC1029 (SEQ ID NO 214) or SEQ ID NO 220 and does notcomprise a 2′-O-Me substituted nucleotide at position 6 to 9, 20, 22, 24to 27, 30 or 32 to 33. In another embodiment, the aptamer of theinvention comprises the nucleic acid sequence of ARC1172 (SEQ ID NO 222)or ARC1115 (SEQ ID NO 221) or ARC1029 (SEQ ID NO 214) or SEQ ID NO 220and comprises a 2′-O-Me substituted nucleotide at one or more positions,at 5 or more positions, at 10 or more positions , at 15 or morepositions, or at 20 or more positions. In another embodiment, theaptamer of the invention comprises the nucleic acid sequence of ARC1172(SEQ ID NO 222) or ARC1115 (SEQ ID NO 221) or ARC1029 (SEQ ID NO 214) orSEQ ID NO 220 and comprises a 2′-O-Me substituted nucleotide at allpositions selected from the group consisting of: position 1 to 5,position 10 to 19, position 21, position 23, position 28 to 29, andposition 34 to 41 wherein the position numbering starts at the 5′ end ofthe nucleic acid sequence.

In a particular embodiment of the invention, an aptamer comprising anucleotide sequence selected from the group consisting of: SEQ ID NO 95to 97 and SEQ ID NO 217 to 219 wherein: Y=C or T/U, R=A or G, N=anynucleotide but in paired regions it assumes a Watson/Crick base pair;and X=1 to 4, is provided. In another embodiment, an aptamer of theinvention is selected from the group consisting of: SEQ ID NO 217 and220 wherein N_(x)-N_(x)-N_(x)-N_(x)-, or N₍₃₋₁₀₎ may be replaced with aPEG linker. In yet another embodiment, an aptamer of the invention isselected from the group consisting of SEQ ID NOs 325-327, where Y=C orT, R=A or G.

In a particular embodiment, the aptamer that binds specifically to vonWillebrand Factor comprises a three way helix junction secondarystructure motif having the consensus sequence structure of SEQ ID NO 220depicted in FIG. 15. In another particular embodiment, the aptamerhaving the three way helix junction comprises the consensus structuredepicted in FIG. 19 A (ARC1368 (SEQ ID NO 291)). While in anotherembodiment, the aptamer having the three way helix junction comprisesthe consensus structure depicted in FIG. 19B (ARC1534 (SEQ ID NO 315)).

In another embodiment, the aptamer that binds specifically to vonWillebrand Factor comprises a stem-loop-stem-loop secondary structuremotif having the consensus sequence structure of SEQ ID NO 217 depictedin FIG. 10. In another embodiment, the aptamer that specifically bindsto von Willebrand Factor comprises the stem-loop-loop secondarystructure motif having the consensus sequence structure of SEQ ID NO 218depicted in FIG. 12. In another embodiment, the aptamer that bindsspecifically to von Willebrand Factor comprises a three way junctionsecondary structure motif with two helical stems and a stem-loop of SEQID NO 19 as depicted in FIG. 13. In some embodiments, the secondarystructure motif of the aptamer of the invention is predicted by:RNAstructure, Version 4.1 (Mathews, D. H.; Disney, M. D.; Childs, J. L.;Schroeder, S. J.; Zuker, M.; and Turner, D. H., “Incorporating chemicalmodification constraints into a dynamic programming algorithm forprediction of RNA secondary structure,” 2004. Proceedings of theNational Academy of Sciences, US, 101, 7287-7292).

In a preferred embodiment, an aptamer that specifically binds to a humanvon Willebrand Factor target and to a non-human von Willebrand Factortarget is provided.

In one embodiment, an aptamer comprising the following structure or asalt thereof is provided:

-   -   wherein: n is about 454 ethylene oxide units (PEG=20 kDa)        is a linker,    -   and the aptamer is an anti-vWF aptamer of the invention. In a        particular embodiment, the aptamer comprises the following        nucleic acid sequence or fragment thereof:        mGmCmGmUdGdCdAmGmUmGmCmCmUmUmCmGmGmCdCmG-s-dTmGdCdGdGTmGmCdCmUdCdCmGmUdCmAmCmGmC-3T        (SEQ ID NO 291) wherein m refers to a 2′-OMe substitution, the        “d” refers to a deoxy nucleotide, the “s” refers to a        phosphorothioate substitution and “3T” refers to an inverted        deoxy thymidine. In another embodiment, the aptamer comprises        the following nucleic acid sequence or fragment thereof:        dGdGdCdGdTdGdCdAdGdTdGdCdCdTdTdCdGdGdCdCdGdTdGdCdGdGdTdGdCdCdTdCdCdGdTdCdAdCdGdCdC-3T        (SEQ ID NO 323) wherein “d” refers to a deoxy nucleotide and        “3T” refers to an inverted deoxy thymidine. In some embodiments        of this aspect of the invention the linker is an alkyl linker.        In particular embodiments, the alkyl linker comprises 2 to 18        consecutive CH₂ groups. In preferred embodiments, the alkyl        linker comprises 2 to 12 consecutive CH₂ groups. In particularly        preferred embodiments the alkyl linker comprises 3 to 6        consecutive CH₂ groups.

In a particular embodiment, the aptamer of the invention comprises thefollowing structure:

wherein: n is about 454 ethylene oxide units (PEG=20 kDa), and theaptamer nucleic acid sequence is an anti-vWF aptamer of the invention.In a particular embodiment, the aptamer comprises the following nucleicacid sequence or fragment thereof:mGmCmGmUdGdCdAmGmUmGmCmCmUmUmCmGmGmCdCmG-s-dTmGdCdGdGTmGmCdCmUdCdCmGmUdCmAmCmGmC-3T(SEQ ID NO 291) wherein m refers to a 2′-OMe substitution, the “d”refers to a deoxy nucleotide, the “s” refers to a phosphorothioatesubstitution and “3T” refers to an inverted deoxy thymidine. In anotherembodiment, the aptamer comprises the following nucleic acid sequence orfragment thereof:dGdGdCdGdTdGdCdAdGdTdGdCdCdTdTdCdGdGdCdCdGdTdGdCdGdGdTdGdCdCdTdCdCdGdTdCdAdCdGdCdC-3T(SEQ ID NO 323) wherein “d” refers to a deoxy nucleotide and “3T” refersto an inverted deoxy thymidine.

In another embodiment, a salt of an aptamer of the invention isprovided. In a particular embodiment, the following salt of an aptameris provided:

-   -   N-(methoxy-polyethyleneglycol)-6-aminohexylyl-(1→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-cytidylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-deoxyguanylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-deoxyadenylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-cytidylyl-(3′→5′)-2′-OMe-cytidylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-OMe-cytidylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-cytidylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-OMe-P-thioguanylyl-(3′→5′)-2′-deoxythymidylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-deoxyguanylyl-(3′→5′)-2′-deoxyguanylyl-(3′→5′)-2′-deoxythymidylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-cytidylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-uracylyl-(3′→5′)-2′-deoxycytidylyl-(3′→5′)-2′-OMe-adenylyl-(3′→5′)-2′-OMe-cytidylyl-(3′→5′)-2′-OMe-guanylyl-(3′→5′)-2′-OMe-cytidylyl-(3′→5′)-(3′→3′)-2′-deoxythymidine,        40-sodium salt, wherein the methoxy polyethyleneglycol comprises        a molecular weight of 20 kDa.

In yet another embodiment, a pharmaceutical composition comprising atherapeutically effective amount of any one of the aptamers of theinvention or a salt thereof and a pharmaceutically acceptable carrier ordiluent is provided. In a particular embodiment, the pharmaceuticalcomposition of the invention comprises ARC1779. In a more particularembodiment the pharmaceutical composition comprises an aptamer havingthe following structure or a salt thereof:

wherein: n is about 454 ethylene oxide units (PEG=20 kDa), and theaptamer comprises the following nucleic acid sequence or fragmentthereof:mGmCmGmUdGdCdAmGmUmGmCmCmUmUmCmGmGmCdCmG-s-dTmGdCdGdGTmGmCdCmUdCdCmGmUdCmAmCmGmC-3T(SEQ ID NO 291) wherein m refers to a 2′-OMe substitution, the “d”refers to a deoxy nucleotide, the “s” refers to a phosphorothioatesubstitution and “3T” refers to an inverted deoxy thymidine.

The invention provides a method of treating, preventing or amelioratinga disease mediated by von Willebrand Factor, comprising administering anaptamer or a pharmaceutical composition of the invention to avertebrate, preferably a mammal, more preferably a human. In someembodiments, the disease to be treated, prevented or ameliorated isselected from the group consisting of: essential thrombocytopenia,thrombotic thrombocopenic purpura (“TTP”), Type IIb von Willebrand'sdisease, pseudo von Willebrand disease, peripheral artery disease, e.g.peripheral arterial occlusive disease, unstable angina, angina pectoris,arterial thrombosis, atherosclerosis, myocardial infarction, acutecoronary syndrome, atrial fibrillation, carotid stenosis, cerebralinfarction, cerebral thrombosis, ischemic stroke, and transient cerebralischemic attack. In some embodiments, the pharmaceutical composition ofthe invention is administered prior to/during and/or after dialysis,CABG surgery, percutaneous coronary intervention or heart valvereplacement.

The length of the in vivo half life of the aptamer of the invention mayvary depending on the disease to be treated. ameliorated and/orprevented. For example, in some embodiments in which chronic aptameradministration is desirable due to the characteristics of the disease tobe treated, ameliorated and/or prevented, the aptamer of the inventionmay comprise a relatively long half life, e.g. a half life greater thanfive hours in humans.

In other embodiments, the aptamer of the invention comprises a desiredfunctional half life or duration of effect. Functional half life orduration of effect is a function of both pharmacokinetic half life andpharmacodynamic activity of the aptamer. In some embodiments, thedesired human functional half-life or duration of effect for an anti-vWFtherapeutic aptamer is on the order of 1-5 hours for the proposedindications elective PCI and ACS. Aptamers with such kinetics representa balance between the dual objectives of (1) minimizing total aptamerdose (achieved with longer half-life) and (2) allowing rapidnormalization of platelet function following cessation of treatment(achieved with shorter half-life). In some embodiments, rapidnormalization of platelet function is important as it allows cliniciansthe option of rapid intervention (e.g. CABG) should a patient fail tostabilize in response to treatment.

Accordingly, in some embodiments the aptamer for use in the methods oftreatment and/or pharmaceutical compositions of the invention comprisesa relatively short functional half life, e.g. a functional half life inhumans of about 1 to 5 hours. In some embodiments, the functional halflife in humans is at least 1 hour, at least 2 hours, at least 3 hours,at least 4 hours and not more than about 5 hours. In some embodiments,the functional half life or duration of effect is about the same as thedistribution half life T_(1/2α) of the aptamer.

In some embodiments, the aptamer of the invention comprising the shortfunctional half life in humans is for use in methods and compositionsfor the treatment, amelioration or prevention of diseases thatpotentially may require surgical intervention, such as acute coronarysyndrome. In some embodiments, the aptamer of this aspect of theinvention comprising a short functional half life in humans isconjugated to a PEG, e.g. a 5, 10 or 20 kDa PEG. In some embodiments,the aptamer of this aspect of the invention comprising a short half lifein humans is ARC1779.

The invention also provides a diagnostic method comprising contacting anaptamer of the invention with a composition suspected of comprising vonWillebrand Factor, von Willebrand Factor domain A1 or a variant thereofand detecting the presence or absence of von Willebrand Factor, vonWillebrand Factor domain A1 or a variant thereof. In some embodiments,the diagnostic method is for use in vitro while in other embodiments,the diagnostic method is for use in vivo.

The invention also provides a method for identifying an aptamer thatblocks a biological function in vivo comprising:

-   -   a) preparing a candidate mixture of single-stranded nucleic        acids;    -   b) contacting the candidate mixture with both a full length        protein target and a domain of the full length protein target;    -   c) partitioning the nucleic acids having an increased affinity        for the full length protein target or the protein target domain;        and    -   d) amplifying the increased affinity nucleic acids, in vitro, to        yield a protein target specific enriched aptamer mixture.

In some embodiments, the identification method further comprises;

-   -   e) contacting the target specific enriched aptamer mixture with        the full length protein target;    -   f) partitioning the nucleic acids having an increased affinity        for the full length protein target; and    -   g) amplifying the increased affinity nucleic acids, in vitro; to        yield a target specific enriched aptamer mixture;    -   h) contacting the target specific enriched aptamer mixture with        the protein target domain;    -   i) partitioning the nucleic acids having an increased affinity        for the protein target domain; and    -   j) amplifying the increased affinity nucleic acids, in vitro, to        yield a protein target specific enriched aptamer mixture.

In some embodiments, the identification method further comprisesselecting an aptamer that blocks a biological function of the fulllength protein target in vivo while in other embodiments, the methodfurther comprises selecting an aptamer that blocks a biological functionof the protein target domain in vivo. In some embodiments of theidentification method of the invention, the full length protein targetis from a first species and the protein target domain is from a secondspecies. In further embodiments of the identification method of theinvention, the method further comprises selecting an aptamer capable ofbinding to both protein targets of both the first and second species,preferably selecting an aptamer that blocks a biological function of theprotein target in both the first and second species. In some embodimentsof the identification method of the invention, the full length targetprotein target is von Willebrand Factor. In some embodiments of theidentification method of the invention, the full length target proteintarget is von Willebrand Factor, wherein it is preferred that theselected aptamer blocks von Willebrand Factor mediated plateletaggregation. In some embodiments the protein target domain is vonWillebrand Factor domain A1. The invention also provides an aptameridentified by the identification method of the invention.

In some embodiments, the invention also provides an aptamer thatspecifically binds to von Willebrand Factor comprising a primary nucleicacid sequence at least 80% identical, particularly at least 90%identical, and more particularly at least 95% identical to any one ofthe primary nucleic acid sequences selected from the group consisting ofSEQ ID NOS 11 to 50, SEQ ID NOS 54 to 94, SEQ ID NOS 98 to 164, SEQ IDNO 165, SEQ ID NO 169, SEQ ID NO 172, SEQ ID NO 174, SEQ ID NO 177, SEQID NO 180, SEQ ID NO 183, SEQ ID NO 186, SEQ ID NO 189, SEQ ID NO 192,SEQ ID NO 198, SEQ ID NO 201, SEQ ID NO 205, SEQ ID NO 208, SEQ ID NOS212-214, ARC1115 (SEQ ID NO 221), ARC1172 (SEQ ID NO 222), ARC1194 (SEQID NO 223) to ARC1240 (SEQ ID NO 269), ARC1338 (SEQ ID NO 273) toARC1346 (SEQ ID NO 281), ARC1361 (SEQ ID NO 284) to ARC1381 (SEQ ID NO304), ARC1524 (SEQ ID NO 305), ARC1526 (SEQ ID NO 307) to ARC1535 (SEQID NO 316), ARC1546 (SEQ ID NO 317), ARC1635 (SEQ ID NO 319), ARC1759(SEQ ID NO 318), ARC1779 to ARC1780 (SEQ ID NO 321) and ARC1884 (SEQ IDNO 322) to ARC1885 (SEQ ID NO 323). In some embodiments, the % sequenceidentity of the aptamers of the invention is BLAST sequence identity.

In another embodiment, the aptamer of the invention comprises a nucleicacid sequence having chemical modifications that including chemicalmodifications is at least 80% identical, particularly 90% identical, andmore particularly at least 95% identical to any one of the nucleic acidsequences selected from the group consisting of: SEQ ID NOS 11 to 50,SEQ ID NOS 54 to 94, SEQ ID NOS 98 to 164, SEQ ID NO 165, SEQ ID NO 169,SEQ ID NO 172, SEQ ID NO 174, SEQ ID NO 177, SEQ ID NO 180, SEQ ID NO183, SEQ ID NO 186, SEQ ID NO 189, SEQ ID NO 192, SEQ ID NO 198, SEQ IDNO 201, SEQ ID NO 205, SEQ ID NO 208, SEQ ID NOS 212-214, ARC1115 (SEQID NO 221), ARC1172 (SEQ ID NO 222), ARC1194 (SEQ ID NO 223) to ARC1240(SEQ ID NO 269), ARC1338 (SEQ ID NO 273) to ARC1346 (SEQ ID NO 281),ARC1361 (SEQ ID NO 284) to ARC1381 (SEQ ID NO 304), ARC1524 (SEQ ID NO305), ARC1526 (SEQ ID NO 307) to ARC1535 (SEQ ID NO 316), ARC1546 (SEQID NO 317), ARC1635 (SEQ ID NO 319), ARC1759 (SEQ ID NO 318), ARC1779 toARC1780 (SEQ ID NO 321) and ARC1884 (SEQ ID NO 322) to ARC1885 (SEQ IDNO 323).

In yet another embodiment, the invention provides an aptamer that uponbinding a von Willebrand Factor target modulates a von Willebrand Factorfunction, preferably in vivo and comprises a sequence of 30 contiguousnucleotides that are identical to a sequence of 30 contiguousnucleotides comprised in any one of the sequences selected from thegroup of: SEQ ID NOS 11 to 50, SEQ ID NOS 54 to 94, SEQ ID NOS 98 to164, SEQ ID NO 165, SEQ ID NO 169, SEQ ID NO 172, SEQ ID NO 174, SEQ IDNO 177, SEQ ID NO 180, SEQ ID NO 183, SEQ ID NO 186, SEQ ID NO 189, SEQID NO 192, SEQ ID NO 198, SEQ ID NO 201, SEQ ID NO 205, SEQ ID NO 208,SEQ ID NOS 212-214, ARC1115 (SEQ ID NO 221), ARC1172 (SEQ ID NO 222),ARC1194 (SEQ ID NO 223) to ARC1240 (SEQ ID NO 269), ARC1338 (SEQ ID NO273) to ARC1346 (SEQ ID NO 281), 1361 (SEQ ID NO 284) to ARC1381 (SEQ IDNO 304), ARC1524 (SEQ ID NO 305), ARC1526 (SEQ ID NO 307) to ARC1535(SEQ ID NO 316), ARC1546 (SEQ ID NO 317), ARC1635 (SEQ ID NO 319),ARC1759 (SEQ ID NO 318), ARC1779 to ARC1780 (SEQ ID NO 321) and ARC1884(SEQ ID NO 322) to ARC1885 (SEQ ID NO 323). In yet another embodiment,the aptamer of the invention upon binding a von Willebrand Factor targetmodulates a von Willebrand Factor function, preferably in vivo, andcomprises 20 contiguous nucleotides that are identical to a sequence of20 contiguous nucleotides in the unique sequence region of any one ofthe aptamer selected from the group of: SEQ ID NOS 11 to 50, SEQ ID NOS54 to 94, SEQ ID NOS 98 to 164, SEQ ID NO 165, SEQ ID NO 169, SEQ ID NO172, SEQ ID NO 174, SEQ ID NO 177, SEQ ID NO 180, SEQ ID NO 183, SEQ IDNO 186, SEQ ID NO 189, SEQ ID NO 192, SEQ ID NO 198, SEQ ID NO 201, SEQID NO 205, SEQ ID NO 208, SEQ ID NOS 212-214, ARC1115 (SEQ ID NO 221),ARC1172 (SEQ ID NO 222), ARC1194 (SEQ ID NO 223) to ARC1240 (SEQ ID NO269), ARC1338 (SEQ ID NO 273) to ARC1346 (SEQ ID NO 281), ARC1361 (SEQID NO 284) to ARC1381 (SEQ ID NO 304), ARC1524 (SEQ ID NO 305), ARC1526(SEQ ID NO 307) to ARC1535 (SEQ ID NO 316), ARC1546 (SEQ ID NO 317),ARC1635 (SEQ ID NO 319), ARC1759 (SEQ ID NO 318), ARC1779 to ARC1780(SEQ ID NO 321) and ARC1884 (SEQ ID NO 322) to ARC1885 (SEQ ID NO 323).In yet another embodiment, the aptamer of the invention upon binding avon Willebrand Factor target modulates a von Willebrand Factor function,preferably in vivo, and comprises 8 contiguous nucleotides that areidentical to a sequence of 8 contiguous nucleotides in the uniquesequence region of any one of the aptamer selected from the group of:SEQ ID NOS 11 to 50, SEQ ID NOS 54 to 94, SEQ ID NOS 98 to 164, SEQ IDNO 165, SEQ ID NO 169, SEQ ID NO 172, SEQ ID NO 174, SEQ ID NO 177, SEQID NO 180, SEQ ID NO 183, SEQ ID NO 186, SEQ ID NO 189, SEQ ID NO 192,SEQ ID NO 198, SEQ ID NO 201, SEQ ID NO 205, SEQ ID NO 208, SEQ ID NOS212-214, ARC1115 (SEQ ID NO 221), ARC1172 (SEQ ID NO 222) (SEQ ID NO222), ARC1194 (SEQ ID NO 223) to ARC1240 (SEQ ID NO 269), ARC1338 (SEQID NO 273) to ARC1346 (SEQ ID NO 281), ARC1361 (SEQ ID NO 284) toARC1381 (SEQ ID NO 304), ARC1524 (SEQ ID NO 305), ARC1526 (SEQ ID NO307) to ARC1535 (SEQ ID NO 316), ARC1546 (SEQ ID NO 317), ARC1635 (SEQID NO 319), ARC1759 (SEQ ID NO 318), ARC1779 to ARC1780 (SEQ ID NO 321)and ARC1884 (SEQ ID NO 322) to ARC1885 (SEQ ID NO 323). In yet anotherembodiment, the aptamer of the invention upon binding a von WillebrandFactor target modulates a von Willebrand Factor function, preferably invivo, and comprises 4 contiguous nucleotides that are identical to asequence of 4 contiguous nucleotides in the unique sequence region ofany one of the aptamer selected from the group of: SEQ ID NOS 11 to 50,SEQ ID NOS 54 to 94, SEQ ID NOS 98 to 164, SEQ ID NO 165, SEQ ID NO 169,SEQ ID NO 172, SEQ ID NO 174, SEQ ID NO 177, SEQ ID NO 180, SEQ ID NO183, SEQ ID NO 186, SEQ ID NO 189, SEQ ID NO 192, SEQ ID NO 198, SEQ IDNO 201, SEQ ID NO 205, SEQ ID NO 208, SEQ ID NOS 212-214, ARC1115 (SEQID NO 221), ARC1172 (SEQ ID NO 222) (SEQ ID NO 222), ARC1194 (SEQ ID NO223) to ARC1240 (SEQ ID NO 269), ARC1338 (SEQ ID NO 273) to ARC1346 (SEQID NO 281), ARC1361 (SEQ ID NO 284) to ARC1381 (SEQ ID NO 304), ARC1524(SEQ ID NO 305), ARC1526 (SEQ ID NO 307) to ARC1535 (SEQ ID NO 316),ARC1546 (SEQ ID NO 317), ARC1635 (SEQ ID NO 319), ARC1759 (SEQ ID NO318), ARC1779 to ARC1780 (SEQ ID NO 321) and ARC1884 (SEQ ID NO 322) toARC1885 (SEQ ID NO 323).

DETAILED DESCRIPTION OF THE INVENTION

The details of one or more embodiments of the invention are set forth inthe accompanying description below. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are now described. Other features, objects, and advantages ofthe invention will be apparent from the description. In thespecification, the singular forms also include the plural unless thecontext clearly dictates otherwise. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. In the case of conflict, the present Specificationwill control.

The SELEX™ Method

A suitable method for generating an aptamer is with the process entitled“Systematic Evolution of Ligands by Exponential Enrichment” (“SELEX™”)generally depicted in FIG. 1. The SELEX™ process is a method for the invitro evolution of nucleic acid molecules with highly specific bindingto target molecules and is described in, e.g., U.S. patent applicationSer. No. 07/536,428, filed Jun. 11, 1990, now abandoned, U.S. Pat. No.5,475,096 entitled “Nucleic Acid Ligands”, and U.S. Pat. No. 5,270,163(see also WO 91/19813) entitled “Nucleic Acid Ligands”. EachSELEX™-identified nucleic acid ligand, i.e., each aptamer, is a specificligand of a given target compound or molecule. The SELEX™ process isbased on the unique insight that nucleic acids have sufficient capacityfor forming a variety of two- and three-dimensional structures andsufficient chemical versatility available within their monomers to actas ligands (i.e., form specific binding pairs) with virtually anychemical compound, whether monomeric or polymeric. Molecules of any sizeor composition can serve as targets.

SELEX™ relies as a starting point upon a large library or pool of singlestranded oligonucleotides comprising randomized sequences. Theoligonucleotides can be modified or unmodified DNA, RNA, or DNA/RNAhybrids. In some examples, the pool comprises 100% random or partiallyrandom oligonucleotides. In other examples, the pool comprises random orpartially random oligonucleotides containing at least one fixed sequenceand/or conserved sequence incorporated within randomized sequence. Inother examples, the pool comprises random or partially randomoligonucleotides containing at least one fixed and/or conserved sequenceat its 5′ and/or 3′ end which may comprise a sequence shared by all themolecules of the oligonucleotide pool. Fixed sequences are sequencescommon to oligonucleotides in the pool which are incorporated for apreselected purpose, such as CpG motifs described further below,hybridization sites for PCR primers, promoter sequences for RNApolymerases (e.g., T3, T4, T7, and SP6), restriction sites, orhomopolymeric sequences, such as poly A or poly T tracts, catalyticcores, sites for selective binding to affinity columns, and othersequences to facilitate cloning and/or sequencing of an oligonucleotideof interest. Conserved sequences are sequences, other than thepreviously described fixed sequences, shared by a number of aptamersthat bind to the same target.

The oligonucleotides of the pool preferably include a randomizedsequence portion as well as fixed sequences necessary for efficientamplification. Typically the oligonucleotides of the starting poolcontain fixed 5′ and 3′ terminal sequences which flank an internalregion of 30-50 random nucleotides. The randomized nucleotides can beproduced in a number of ways including chemical synthesis and sizeselection from randomly cleaved cellular nucleic acids. Sequencevariation in test nucleic acids can also be introduced or increased bymutagenesis before or during the selection/amplification iterations.

The random sequence portion of the oligonucleotide can be of any lengthand can comprise ribonucleotides and/or deoxyribonucleotides and caninclude modified or non-natural nucleotides or nucleotide analogs. See,e.g., U.S. Pat. Nos. 5,958,691; 5,660,985; 5,958,691; 5,698,687;5,817,635; 5,672,695, and PCT Publication WO 92/07065. Randomoligonucleotides can be synthesized from phosphodiester-linkednucleotides using solid phase oligonucleotide synthesis techniques wellknown in the art. See, e.g., Froehler et al., Nucl. Acid Res.14:5399-5467 (1986) and Froehler et al., Tet. Lett. 27:5575-5578 (1986).Random oligonucleotides can also be synthesized using solution phasemethods such as triester synthesis methods. See, e.g., Sood et al.,Nucl. Acid Res. 4:2557 (1977) and Hirose et al., Tet. Lett., 28:2449(1978). Typical syntheses carried out on automated DNA synthesisequipment yield 10¹⁴-10¹⁶ individual molecules, a number sufficient formost SELEX™ experiments. Sufficiently large regions of random sequencein the sequence design increases the likelihood that each synthesizedmolecule is likely to represent a unique sequence.

The starting library of oligonucleotides may be generated by automatedchemical synthesis on a DNA synthesizer. To synthesize randomizedsequences, mixtures of all four nucleotides are added at each nucleotideaddition step during the synthesis process, allowing for randomincorporation of nucleotides. As stated above, in one embodiment, randomoligonucleotides comprise entirely random sequences; however, in otherembodiments, random oligonucleotides can comprise stretches of nonrandomor partially random sequences. Partially random sequences can be createdby adding the four nucleotides in different molar ratios at eachaddition step.

The starting library of oligonucleotides may be either RNA or DNA, orsubstituted RNA or DNA. In those instances where an RNA library is to beused as the starting library it is typically generated by synthesizing aDNA library, optionally PCR amplifying, then transcribing the DNAlibrary in vitro using T7 RNA polymerase or modified T7 RNA polymerases,and purifying the transcribed library. The RNA or DNA library is thenmixed with the target under conditions favorable for binding andsubjected to step-wise iterations of binding, partitioning andamplification, using the same general selection scheme, to achievevirtually any desired criterion of binding affinity and selectivity.More specifically, starting with a mixture containing the starting poolof nucleic acids, the SELEX™ method includes steps of: (a) contactingthe mixture with the target under conditions favorable for binding; (b)partitioning unbound nucleic acids from those nucleic acids which havebound specifically to target molecules; (c) dissociating the nucleicacid-target complexes; (d) amplifying the nucleic acids dissociated fromthe nucleic acid-target complexes to yield a ligand-enriched mixture ofnucleic acids; and (e) reiterating the steps of binding, partitioning,dissociating and amplifying through as many cycles as desired to yieldhighly specific, high affinity nucleic acid ligands to the targetmolecule. In those instances where RNA aptamers are being selected, theSELEX™ method further comprises the steps of: (i) reverse transcribingthe nucleic acids dissociated from the nucleic acid-target complexesbefore amplification in step (d); and (ii) transcribing the amplifiednucleic acids from step (d) before restarting the process.

Within a nucleic acid mixture containing a large number of possiblesequences and structures, there is a wide range of binding affinitiesfor a given target. A nucleic acid mixture comprising, for example, a 20nucleotide randomized segment can have 4²⁰ candidate possibilities.Those which have the higher affinity (lower dissociation constants) forthe target are most likely to bind to the target. After partitioning,dissociation and amplification, a second nucleic acid mixture isgenerated, enriched for the higher binding affinity candidates.Additional rounds of selection progressively favor the best ligandsuntil the resulting nucleic acid mixture is predominantly composed ofonly one or a few sequences. These can then be cloned, sequenced andindividually tested for binding affinity as pure ligands or aptamers.

Cycles of selection and amplification are repeated until a desired goalis achieved. In the most general case, selection/amplification iscontinued until no significant improvement in binding strength isachieved on repetition of the cycle. The method is typically used tosample approximately 10¹⁴ different nucleic acid species but may be usedto sample as many as about 10¹⁸ different nucleic acid species.Generally, nucleic acid aptamer molecules are selected in a 5 to 20cycle procedure. In one embodiment, heterogeneity is introduced only inthe initial selection stages and does not occur throughout thereplicating process.

In one embodiment of SELEX™, the selection process is so efficient atisolating those nucleic acid ligands that bind most strongly to theselected target, that only one cycle of selection and amplification isrequired. Such an efficient selection may occur, for example, in achromatographic-type process wherein the ability of nucleic acids toassociate with targets bound on a column operates in such a manner thatthe column is sufficiently able to allow separation and isolation of thehighest affinity nucleic acid ligands.

In many cases, it is not necessarily desirable to perform the iterativesteps of SELEX™ until a single nucleic acid ligand is identified. Thetarget-specific nucleic acid ligand solution may include a family ofnucleic acid structures or motifs that have a number of conservedsequences and a number of sequences which can be substituted or addedwithout significantly affecting the affinity of the nucleic acid ligandsto the target. By terminating the SELEX™ process prior to completion, itis possible to determine the sequence of a number of members of thenucleic acid ligand solution family.

A variety of nucleic acid primary, secondary and tertiary structures areknown to exist. The structures or motifs that have been shown mostcommonly to be involved in non-Watson-Crick type interactions arereferred to as hairpin loops, symmetric and asymmetric bulges,pseudoknots and myriad combinations of the same. Almost all known casesof such motifs suggest that they can be formed in a nucleic acidsequence of no more than 30 nucleotides. For this reason, it is oftenpreferred that SELEX™ procedures with contiguous randomized segments beinitiated with nucleic acid sequences containing a randomized segment ofbetween about 20 to about 50 nucleotides, and of about 30 to about 40nucleotides in some embodiments. In one example, the5′-fixed:random:3′-fixed sequence comprises a random sequence of about30 to about 50 nucleotides.

The core SELEX™ method has been modified to achieve a number of specificobjectives. For example, U.S. Pat. No. 5,707,796 describes the use ofSELEX™ in conjunction with gel electrophoresis to select nucleic acidmolecules with specific structural characteristics, such as bent DNA.U.S. Pat. No. 5,763,177 describes SELEX™ based methods for selectingnucleic acid ligands containing photoreactive groups capable of bindingand/or photocrosslinking to and/or photoinactivating a target molecule.U.S. Pat. Nos. 5,567,588 and 5,861,254 describe SELEX™ based methodswhich achieve highly efficient partitioning between oligonucleotideshaving high and low affinity for a target molecule. U.S. Pat. No.5,496,938 describes methods for obtaining improved nucleic acid ligandsafter the SELEX™ process has been performed. U.S. Pat. No. 5,705,337describes methods for covalently linking a ligand to its target.

SELEX™ can also be used to obtain nucleic acid ligands that bind to morethan one site on the target molecule, and to obtain nucleic acid ligandsthat include non-nucleic acid species that bind to specific sites on thetarget. SELEX™ provides means for isolating and identifying nucleic acidligands which bind to any envisionable target, including large and smallbiomolecules such as nucleic acid-binding proteins and proteins notknown to bind nucleic acids as part of their biological function as wellas cofactors and other small molecules. For example, U.S. Pat. No.5,580,737 discloses nucleic acid sequences identified through SELEX™which are capable of binding with high affinity to caffeine and theclosely related analog, theophylline.

Counter-SELEX™ is a method for improving the specificity of nucleic acidligands to a target molecule by eliminating nucleic acid ligandsequences with cross-reactivity to one or more non-target molecules.Counter-SELEX™ is comprised of the steps of: (a) preparing a candidatemixture of nucleic acids; (b) contacting the candidate mixture with thetarget, wherein nucleic acids having an increased affinity to the targetrelative to the candidate mixture may be partitioned from the remainderof the candidate mixture; (c) partitioning the increased affinitynucleic acids from the remainder of the candidate mixture; (d)dissociating the increased affinity nucleic acids from the target; (e)contacting the increased affinity nucleic acids with one or morenon-target molecules such that nucleic acid ligands with specificaffinity for the non-target molecule(s) are removed; and (f) amplifyingthe nucleic acids with specific affinity only to the target molecule toyield a mixture of nucleic acids enriched for nucleic acid sequenceswith a relatively higher affinity and specificity for binding to thetarget molecule. As described above for SELEX™ cycles of selection andamplification are repeated as necessary until a desired goal isachieved.

One potential problem encountered in the use of nucleic acids astherapeutics and vaccines is that oligonucleotides in theirphosphodiester form may be quickly degraded in body fluids byintracellular and extracellular enzymes such as endonucleases andexonucleases before the desired effect is manifest. The SELEX™ methodthus encompasses the identification of high-affinity nucleic acidligands containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the sugar and/or phosphate and/or basepositions. SELEX™—identified nucleic acid ligands containing modifiednucleotides are described, e.g., in U.S. Pat. No. 5,660,985, whichdescribes oligonucleotides containing nucleotide derivatives chemicallymodified at the 2′ position of ribose, 5 position of pyrimidines, and 8position of purines, U.S. Pat. No. 5,756,703 which describesoligonucleotides containing various 2′-modified pyrimidines, and U.S.Pat. No. 5,580,737 which describes highly specific nucleic acid ligandscontaining one or more nucleotides modified with 2′-amino (2′-NH₂),2′-fluoro (2′-F), and/or 2′-OMe substituents.

Modifications of the nucleic acid ligands contemplated in this inventioninclude, but are not limited to, those which provide other chemicalgroups that incorporate additional charge, polarizability,hydrophobicity, hydrogen bonding, electrostatic interaction, andfluxionality to the nucleic acid ligand bases or to the nucleic acidligand as a whole. Modifications to generate oligonucleotide populationswhich are resistant to nucleases can also include one or more substituteinternucleotide linkages, altered sugars, altered bases, or combinationsthereof. Such modifications include, but are not limited to, 2′-positionsugar modifications, 5-position pyrimidine modifications, 8-positionpurine modifications, modifications at exocyclic amines, substitution of4-thiouridine, substitution of 5-bromo or 5-iodo-uracil, backbonemodifications, phosphorothioate or alkyl phosphate modifications,methylations, and unusual base-pairing combinations such as the isobasesisocytidine and isoguanidine. Modifications can also include 3′ and 5′modifications such as capping.

In one embodiment, oligonucleotides are provided in which the P(O)Ogroup is replaced by P(O)S (“thioate”), P(S)S (“dithioate”), P(O)NR₂(“amidate”), P(O)R, P(O)OR′, CO or CH₂ (“formacetal”) or 3′-amine(—NH—CH₂—CH₂—), wherein each R or R′is independently H or substituted orunsubstituted alkyl. Linkage groups can be attached to adjacentnucleotides through an —O—, —N—, or —S— linkage. Not all linkages in theoligonucleotide are required to be identical. As used herein, the termphosphorothioate encompasses one or more non-bridging oxygen atoms in aphosphodiester bond replaced by one or more sulfur atoms

In further embodiments, the oligonucleotides comprise modified sugargroups, for example, one or more of the hydroxyl groups is replaced withhalogen, aliphatic groups, or functionalized as ethers or amines. In oneembodiment, the 2′-position of the furanose residue is substituted byany of an O-methyl, O-alkyl, O-allyl, S-alkyl, S-allyl, or halo group.Methods of synthesis of 2′-modified sugars are described, e.g., inSproat, et al., Nucl. Acid Res. 19:733-738 (1991); Cotten, et al., Nucl.Acid Res. 19:2629-2635 (1991); and Hobbs, et al., Biochemistry12:5138-5145 (1973). Other modifications are known to one of ordinaryskill in the art. Such modifications may be pre-SELEX™ processmodifications or post-SELEX™ process modifications (modification ofpreviously identified unmodified ligands) or may be made byincorporation into the SELEX™ process.

Pre-SELEX™ process modifications or those made by incorporation into theSELEX process yield nucleic acid ligands with both specificity for theirSELEX™ target and improved stability, e.g., in vivo stability.Post-SELEX™ process modifications made to nucleic acid ligands mayresult in improved stability, e.g., in vivo stability without adverselyaffecting the binding capacity of the nucleic acid ligand.

The SELEX™ method encompasses combining selected oligonucleotides withother selected oligonucleotides and non-oligonucleotide functional unitsas described in U.S. Pat. Nos. 5,637,459 and 5,683,867. The SELEX™method further encompasses combining selected nucleic acid ligands withlipophilic or non-immunogenic high molecular weight compounds in adiagnostic or therapeutic complex, as described, e.g., in U.S. Pat. Nos.6,011,020, 6,051,698, and PCT Publication No. WO 98/18480. These patentsand applications teach the combination of a broad array of shapes andother properties, with the efficient amplification and replicationproperties of oligonucleotides, and with the desirable properties ofother molecules.

The identification of nucleic acid ligands to small, flexible peptidesvia the SELEX™ method has also been explored. Small peptides haveflexible structures and usually exist in solution in an equilibrium ofmultiple conformers, and thus it was initially thought that bindingaffinities may be limited by the conformational entropy lost uponbinding a flexible peptide. However, the feasibility of identifyingnucleic acid ligands to small peptides in solution was demonstrated inU.S. Pat. No. 5,648,214 in which high affinity RNA nucleic acid ligandsto substance P, an 11 amino acid peptide, were identified.

The aptamers with specificity and binding affinity to the target(s) ofthe present invention are typically selected by the SELEX™ process asdescribed herein. As part of the SELEX™ process, the sequences selectedto bind to the target are then optionally minimized to determine theminimal sequence having the desired binding affinity. The selectedaptamer sequences and/or the minimized aptamer sequences are optionallyoptimized by performing random or directed mutagenesis of the sequenceto increase binding affinity or alternatively to determine whichpositions in the sequence are essential for binding activity. Forexample, a “doped reselections” may be used to explore the sequencerequirements within an aptamer. During doped reselection, selections arecarried out with a synthetic, degenerate pool that has been designedbased on a single sequence. The level of degeneracy usually varies from70% to 85% wild type nucleotide. In general, neutral mutations areobserved following doped reselection but in some cases sequence changescan result in improvements in affinity. Additionally, selections can beperformed with sequences incorporating modified sequences to stabilizethe aptamer molecules against degradation in vivo.

2′Modified SELEX™

In order for an aptamer to be suitable for use as a therapeutic, it ispreferably inexpensive to synthesize, safe and stable in vivo. Wild-typeRNA and DNA aptamers are typically not stable in vivo because of theirsusceptibility to degradation by nucleases. Resistance to nucleasedegradation can be greatly increased, if desired, by the incorporationof modifying groups at the 2′-position.

2′-fluoro and 2′-amino groups have been successfully incorporated intooligonucleotide libraries from which aptamers have been subsequentlyselected. However, these modifications greatly increase the cost ofsynthesis of the resultant aptamer, and may introduce safety concerns insome cases because of the possibility that the modified nucleotidescould be recycled into host DNA by degradation of the modifiedoligonucleotides and subsequent use of the nucleotides as substrates forDNA synthesis.

Aptamers that contain 2′-O-methyl (“2′-OMe”) nucleotides, as provided insome embodiments herein, overcome many of these drawbacks.Oligonucleotides containing 2′-OMe nucleotides are nuclease-resistantand inexpensive to synthesize. Although 2′-OMe nucleotides areubiquitous in biological systems, natural polymerases do not accept2′-OMe NTPs as substrates under physiological conditions, thus there areno safety concerns over the recycling of 2′-OMe nucleotides into hostDNA. The SELEX™ methods used to generate 2′-modified aptamers isdescribed, e.g., in U.S. Provisional Patent Application Ser. No.60/430,761, filed Dec. 3, 2002, U.S. Provisional Patent Application Ser.No. 60/487,474, filed Jul. 15, 2003, U.S. Provisional Patent ApplicationSer. No. 60/517,039, filed Nov. 4, 2003, U.S. patent application Ser.No. 10/729,581, filed Dec. 3, 2003, U.S. patent application Ser. No.10/873,856 filed Jun. 21, 2004, entitled “Method for in vitro Selectionof 2′-OMe Substituted Nucleic Acids”, and U.S. Provisional PatentApplication Ser. No. 60/696,295, filed Jun. 30, 2005, entitled “ImprovedMaterials and Methods for the Generation of Fully 2′-Modified ContainingNucleic Acid Transcripts”, each of which is herein incorporated byreference in its entirety.

The present invention includes aptamers that bind to and modulate thefunction of von Willebrand Factor which contain modified nucleotides(e.g., nucleotides which have a modification at the 2′ position) to makethe oligonucleotide more stable than the unmodified oligonucleotide toenzymatic and chemical degradation as well as thermal and physicaldegradation. Although there are several examples of 2′-OMe containingaptamers in the literature (see, e.g., Ruckman et al., J.Biol.Chem, 1998273, 20556-20567-695) these were generated by the in vitro selection oflibraries of modified transcripts in which the C and U residues were2′-fluoro (2′-F) substituted and the A and G residues were 2′-OH. Oncefunctional sequences were identified then each A and G residue wastested for tolerance to 2′-OMe substitution, and the aptamer wasre-synthesized having all A and G residues which tolerated 2′-OMesubstitution as 2′-OMe residues. Most of the A and G residues ofaptamers generated in this two-step fashion tolerate substitution with2′-OMe residues, although, on average, approximately 20% do not.Consequently, aptamers generated using this method tend to contain fromtwo to four 2′-OH residues, and stability and cost of synthesis arecompromised as a result. By incorporating modified nucleotides into thetranscription reaction which generate stabilized oligonucleotides usedin oligonucleotide libraries from which aptamers are selected andenriched by SELEX™ (and/or any of its variations and improvements,including those described herein), the methods of the present inventioneliminate the need for stabilizing the selected aptamer oligonucleotides(e.g., by resynthesizing the aptamer oligonucleotides with modifiednucleotides).

In one embodiment, the present invention provides aptamers comprisingcombinations of 2′-OH, 2′-F, 2′-deoxy, and 2′-OMe modifications of theATP, GTP, CTP, TTP, and UTP nucleotides. In another embodiment, thepresent invention provides aptamers comprising combinations of 2′-OH,2′-F, 2′-deoxy, 2′-OMe, 2′-NH₂, and 2′-methoxyethyl modifications of theATP, GTP, CTP, TTP, and UTP nucleotides. In another embodiment, thepresent invention provides aptamers comprising 5⁶ combinations of 2′-OH,2′-F, 2′-deoxy, 2′-OMe, 2′-NH₂, and 2′-methoxyethyl modifications of theATP, GTP, CTP, TTP, and UTP nucleotides.

2′-modified aptamers of some embodiments of the invention are createdusing modified polymerases, e.g., a modified T7 polymerase, having arate of incorporation of modified nucleotides having bulky substituentsat the furanose 2′ position that is higher than that of wild-typepolymerases. For example, a single mutant T7 polymerase (Y639F) in whichthe tyrosine residue at position 639 has been changed to phenylalaninereadily utilizes 2′deoxy, 2′amino-, and 2′fluoro-nucleotidetriphosphates (NTPs) as substrates and has been widely used tosynthesize modified RNAs for a variety of applications. However, thismutant T7 polymerase reportedly can not readily utilize (i.e.,incorporate) NTPs with bulky 2′-substituents such as 2′-OMe or 2′-azido(2′-N₃) substituents. For incorporation of bulky 2′ substituents, adouble T7 polymerase mutant (Y639F/H784A) having the histidine atposition 784 changed to an alanine residue in addition to the Y639Fmutation has been described and has been used in limited circumstancesto incorporate modified pyrimidine NTPs. See Padilla, R. and Sousa, R.,Nucleic Acids Res., 2002, 30(24): 138. A Y639F/H784A/K378R mutant T7 RNApolymerase has been used in limited circumstances to incorporatemodified purine and pyrimidine NTPs, e.g., 2′-OMe NPTs, but requires aspike of 2′-OH GTP for transcription. See Burmeister et. al., Chemistryand Biology, 2005, 12: 25-33. A single mutant T7 polymerase (H784A)having the histidine at position 784 changed to an alanine residue hasalso been described. Padilla et al., Nucleic Acids Research, 2002, 30:138. In both the Y639F/H784A double mutant and H784A single mutant T7polymerases, the change to a smaller amino acid residue such as alanineallows for the incorporation of bulkier nucleotide substrates, e.g.,2′-O methyl substituted nucleotides. See Chelliserry, K. and Ellington,A. D., Nature Biotech, 2004, 9:1155-60. Additional T7 RNA polymerasehave been described with mutations in the active site of the T7 RNApolymerase which more readily incorporate bulky 2′-modified substrates,e.g. a single T7 mutant RNA polymerase having the tyrosine residue atposition 639 changed to a leucine (Y639L). However activity is oftensacrificed for increased substrate specificity conferred by suchmutations, leading to low transcript yields. See Padilla R and Sousa,R., Nucleic Acids Res., 1999, 27(6): 1561.

Generally, it has been found that under the conditions disclosed herein,the Y693F single mutant can be used for the incorporation of all 2′-OMesubstituted NTPs except GTP and the Y639F/H784A, Y639F/H784A/K378R,Y639L/H784A, and Y639L/H784A/K378R mutant T7 RNA polymerases can be usedfor the incorporation of all 2′-OMe substituted NTPs including GTP. Itis expected that the H784A single mutant possesses properties similar tothe Y639F and the Y639F/H784A mutants when used under the conditionsdisclosed herein.

2′-modified oligonucleotides may be synthesized entirely of modifiednucleotides, or with a subset of modified nucleotides. All nucleotidesmay be modified, and all may contain the same modification. Allnucleotides may be modified, but contain different modifications, e.g.,all nucleotides containing the same base may have one type ofmodification, while nucleotides containing other bases may havedifferent types of modification. All purine nucleotides may have onetype of modification (or are unmodified), while all pyrimidinenucleotides have another, different type of modification (or areunmodified). In this way, transcripts, or libraries of transcripts aregenerated using any combination of modifications, including for example,ribonucleotides (2′-OH), deoxyribonucleotides (2′-deoxy), 2′-F, and2′-OMe nucleotides. A transcription mixture containing 2′-OMe C and Uand 2′-OH A and G is referred to as a “rRmY” mixture and aptamersselected therefrom are referred to as “rRmY” aptamers. A transcriptionmixture containing deoxy A and G and 2′-OMe U and C is referred to as a“dRmY” mixture and aptamers selected therefrom are referred to as “dRmY”aptamers. A transcription mixture containing 2′-OMe A, C, and U, and2′-OH G is referred to as a “rGmH” mixture and aptamers selectedtherefrom are referred to as “rGmH” aptamers. A transcription mixturealternately containing 2′-OMe A, C, U and G and 2′-OMe A, U and C and2′-F G is referred to as a “alternating mixture” and aptamers selectedtherefrom are referred to as “alternating mixture” aptamers. Atranscription mixture containing 2′-OMe A, U, C, and G, where up to 10%of the G's are ribonucleotides is referred to as a “r/mGmH” mixture andaptamers selected therefrom are referred to as “r/mGmH” aptamers. Atranscription mixture containing 2′-OMe A, U, and C, and 2′-F G isreferred to as a “fGmH” mixture and aptamers selected therefrom arereferred to as “fGmH” aptamers. A transcription mixture containing2′-OMe A, U, and C, and deoxy G is referred to as a “dGmH” mixture andaptamers selected therefrom are referred to as “dGmH” aptamers. Atranscription mixture containing deoxy A, and 2′-OMe C, G and U isreferred to as a “dAmB” mixture and aptamers selected therefrom arereferred to as “dAmB” aptamers, and a transcription mixture containingall 2′-OH nucleotides is referred to as a “rN” mixture and aptamersselected therefrom are referred to as “rN”, “rRrY”, or “RNA” aptamers. Atranscription mixture containing 2′-OH adenosine triphosphate andguanosine triphosphate and deoxy cytidine triphosphate and thymidinetriphosphate is referred to as a rRdY mixture and aptamers selectedtherefrom are referred to as “rRdY” aptamers. A “mRmY” aptamer is onecontaining only 2′-OMe nucleotides except for the starting nucleotidewhich is 2′-hydroxy.

A preferred embodiment includes any combination of 2′-OH, 2′-deoxy and2′-OMe nucleotides. Another embodiment includes any combination of2′-deoxy and 2′-OMe nucleotides. Yet another embodiment includes anycombination of 2′-deoxy and 2′-OMe nucleotides in which the pyrimidinesare 2′-OMe (such as dRmY, mRmY or dGmH).

Incorporation of modified nucleotides into the aptamers of the inventionmay be accomplished before (pre-) the selection process (e.g., apre-SELEX™ process modification). Optionally, aptamers of the inventionin which modified nucleotides have been incorporated by pre-SELEX™process modification can be further modified by a post-SELEX™modification process (i.e., a post-SELEX™ process modification after apre-SELEX™ modification). Pre-SELEX™ process modifications yieldmodified nucleic acid ligands with specificity for the SELEX™ target andalso improved in vivo stability. Post-SELEX™ process modifications,i.e., modification (e.g., truncation, deletion, substitution oradditional nucleotide modifications of previously identified ligandshaving nucleotides incorporated by pre-SELEX™ process modification) canresult in a further improvement of in vivo stability without adverselyaffecting the binding capacity of the nucleic acid ligand havingnucleotides incorporated by pre-SELEX™™ process modification.

To generate pools of 2′-modified (e.g., 2′-OMe) RNA transcripts inconditions under which a polymerase accepts 2′-modified NTPs the Y693F,Y693F/ K378R, Y693F/H784A, Y693F/H784A/K378R, Y693L/H784A,Y693L/H784A/K378R Y639L, or the Y639L/K378Rmutant T7 RNA polymerases canbe used. A preferred polymerase is the Y639L/H784A mutant T7 RNApolymerase. Another preferred polymerase is the Y639L/H784A/K378R mutantT7 RNA polymerase. Other T7 RNA polymerases, particularly those thatexhibit a high tolerance for bulky 2′-substituents, may also be used inthe present invention. When used in a template-directed polymerizationusing the conditions disclosed herein, the Y639L/H784A or theY639L/H784A/K378R mutant T7 RNA polymerase can be used for theincorporation of all 2′-OMe NTPs, including GTP, with higher transcriptyields than achieved by using the Y639F, Y639F/K378R, Y639F/H784A,Y639F/H784A/K378R, Y639L, or the Y639L/K378R mutant T7 RNA polymerases.The Y639L/H784A and Y639L/H784A/K378R mutant T7 RNA polymerases can beused with but does not require 2′-OH GTP to achieve high yields of2′-modified, e.g., 2′-OMe containing oligonucleotides.

A number of factors have been determined to be important for thetranscription conditions useful in the methods disclosed herein. Forexample, increases in the yields of modified transcript are observedwhen a leader sequence is incorporated into the 5′ end of the DNAtranscription template. The leader sequence is typically 6-15nucleotides long, and may be composed of all purines, or a mixture ofpurine and pyrimidine nucleotides.

Transcription can be divided into two phases: the first phase isinitiation, during which an NTP is added to the 3′-hydroxyl end of GTP(or another substituted guanosine) to yield a dinucleotide which is thenextended by about 10-12 nucleotides; the second phase is elongation,during which transcription proceeds beyond the addition of the firstabout 10-12 nucleotides. It has been found that small amounts of 2′-OHGTP added to a transcription mixture containing an excess of 2′-OMe GTPare sufficient to enable the polymerase to initiate transcription using2′-OH GTP, but once transcription enters the elongation phase thereduced discrimination between 2′-OMe and 2′-OH GTP, and the excess of2′-OMe GTP over 2′-OH GTP allows the incorporation of principally the2′-OMe GTP.

Another important factor in the incorporation of 2′-OMe substitutednucleotides into transcripts is the use of both divalent magnesium andmanganese in the transcription mixture. Different combinations ofconcentrations of magnesium chloride and manganese chloride have beenfound to affect yields of 2′-O-methylated transcripts, the optimumconcentration of the magnesium and manganese chloride being dependent onthe concentration in the transcription reaction mixture of NTPs whichcomplex divalent metal ions. To obtain the greatest yields of maximally2′-O-methylated transcripts (i.e., all 2′-OMe A, C, and U and about 90%of G nucleotides), concentrations of approximately 5 mM magnesiumchloride and 1.5 mM manganese chloride are preferred when each NTP ispresent at a concentration of 0.5 mM. When the concentration of each NTPis 1.0 mM, concentrations of approximately 6.5 mM magnesium chloride and2.0 mM manganese chloride are preferred. When the concentration of eachNTP is 2.0 mM, concentrations of approximately 9.5 mM magnesium chlorideand 3.0 mM manganese chloride are preferred. In any case, departuresfrom these concentrations of up to two-fold still give significantamounts of modified transcripts.

Priming transcription with GMP or guanosine, or another non-2′-OMenon-triphosphate is also important. This effect results from thespecificity of the polymerase for the initiating nucleotide. As aresult, the 5′-terminal nucleotide of any transcript generated in thisfashion is likely to be 2′-OH G. The preferred concentration of GMP (orguanosine) is 0.5 mM and even more preferably 1 mM. It has also beenfound that including PEG, preferably PEG-8000, in the transcriptionreaction is useful to maximize incorporation of modified nucleotides.

For maximum incorporation of 2′-OMe ATP (100%), UTP (100%), CTP(100%)and GTP (˜90%) (“r/mGmH”) into transcripts the following conditions arepreferred: HEPES buffer 200 mM, DTT 40 mM, spermidine 2 mM, PEG-8000 10%(w/v), Triton X-100 0.01% (w/v), MgCl₂ 5 mM (6.5 mM where theconcentration of each 2′-OMe NTP is 1.0 mM), MnCl₂ 1.5 mM (2.0 mM wherethe concentration of each 2′-OMe NTP is 1.0 mM), 2′-OMe NTP (each) 500μM (more preferably, 1.0 mM), 2′-OH GTP 30 μM, 2′-OH GMP 500 μM, pH 7.5,Y639F/H784A T7 RNA Polymerase 200 nM, inorganic pyrophosphatase 5units/ml, and an all-purine leader sequence of at least 8 nucleotideslong. As used herein, one unit of the Y639F/H784A mutant T7 RNApolymerase (or any other mutant T7 RNA polymerase specified herein) isdefined as the amount of enzyme required to incorporate 1 nmole of2′-OMe NTPs into transcripts under the r/mGmH conditions. As usedherein, one unit of inorganic pyrophosphatase is defined as the amountof enzyme that will liberate 1.0 mole of inorganic orthophosphate perminute at pH 7.2 and 25° C.

For maximum incorporation (100%) of 2′-OMe ATP, UTP and CTP (“rGmH”)into transcripts the following conditions are preferred: HEPES buffer200 mM, DTT 40 mM, spermidine 2 mM, PEG-8000 10% (w/v), Triton X-1000.01% (w/v), MgCl₂ 5 mM (9.5 mM where the concentration of each 2′-OMeNTP is 2.0 mM), MnCl₂ 1.5 mM (3.0 mM where the concentration of each2′-OMe NTP is 2.0 mM), 2′-OMe NTP (each) 500 μM (more preferably, 2.0mM), pH 7.5, Y639F T7 RNA Polymerase 200 nM, inorganic pyrophosphatase 5units/ml, and an all-purine leader sequence of at least 8 nucleotideslong.

For maximum incorporation of 2′-OMe ATP (100%), 2′-OMe UTP (100%),2′-OMe CTP (100%) and 2′-OMe GTP (100%) (“mRmY”) into transcripts thefollowing conditions are preferred: HEPES buffer 200 mM, DTT 40 mM,spermidine 2 mM, PEG-8000 10% (w/v), Triton X-100 0.01% (w/v), MgCl₂ 8mM, MnCl₂2.5 mM, 2′-OMe NTP (each) 1.5 mM, 2′-OH GMP 1 mM, pH 7.5,Y639L/H784A/K378R mutant T7 RNA Polymerase 200 nM, inorganicpyrophosphatase 5 units/ml, and a leader sequence that increases thetranscription yield under the derived transcription conditions. In oneembodiment, the leader sequence is an all purine leader sequence. Inanother embodiment, the leader sequence is a mixture of purines andpyrimidines. As used herein, one unit of inorganic pyrophosphatase isdefined as the amount of enzyme that will liberate 1.0 mole of inorganicorthophosphate per minute at pH 7.2 and 25° C.

For maximum incorporation (100%) of 2′-OMe UTP and CTP (“rRmY”) intotranscripts the following conditions are preferred: HEPES buffer 200 mM,DTT 40 mM, spermidine 2 mM, PEG-8000 10% (w/v), Triton X-100 0.01%(w/v), MgCl₂ 5 mM (9.5 mM where the concentration of each 2′-OMe NTP is2.0 mM), MnCl₂ 1.5 mM (3.0 mM where the concentration of each 2′-OMe NTPis 2.0 mM), 2′-OMe NTP (each) 500 μM (more preferably, 2.0 mM), pH 7.5,Y639F/H784A T7 RNA Polymerase 200 nM, inorganic pyrophosphatase 5units/ml, and an all-purine leader sequence of at least 8 nucleotideslong.

For maximum incorporation (100%) of deoxy ATP and GTP and 2′-OMe UTP andCTP (“dRmY”) into transcripts the following conditions are preferred:HEPES buffer 200 mM, DTT 40 mM, spermidine 2 mM, spermidine 2 mM,PEG-8000 10% (w/v), Triton X-100 0.01% (w/v), MgCl₂ 9.5 mM, MnCl₂ 3.0mM, 2′-OMe NTP (each) 2.0 mM, pH 7.5, Y639F T7 RNA Polymerase 200 nM,inorganic pyrophosphatase 5 units/ml, and an all-purine leader sequenceof at least 8 nucleotides long.

For maximum incorporation (100%) of 2′-OMe ATP, UTP and CTP and 2′-F GTP(“fGmH”) into transcripts the following conditions are preferred: HEPESbuffer 200 mM, DTT 40 mM, spermidine 2 mM, PEG-8000 10% (w/v), TritonX-100 0.01% (w/v), MgCl₂ 9.5 mM, MnCl₂ 3.0 mM, 2′-OMe NTP (each) 2.0 mM,pH 7.5, Y639F T7 RNA Polymerase 200 nM, inorganic pyrophosphatase 5units/ml, and an all-purine leader sequence of at least 8 nucleotideslong.

For maximum incorporation (100%) of deoxy ATP and 2′-OMe UTP, GTP andCTP (“dAmB”) into transcripts the following conditions are preferred:HEPES buffer 200 mM, DTT 40 mM, spermidine 2 mM, PEG-8000 10% (w/v),Triton X-100 0.01% (w/v), MgCl₂ 9.5 mM, MnCl₂ 3.0 mM, 2′-OMe NTP (each)2.0 mM, pH 7.5, Y639F T7 RNA Polymerase 200 nM, inorganicpyrophosphatase 5 units/ml, and an all-purine leader sequence of atleast 8 nucleotides long.

For each of the above (a) transcription is preferably performed at atemperature of from about 20° C. to about 50° C., preferably from about30° C. to 45° C., and more preferably about 37° C. for a period of atleast two hours and (b) 50-300 nM of a double stranded DNA transcriptiontemplate is used (200 nM template is used in round 1 to increasediversity (300 nM template is used in dRmY transcriptions)), and forsubsequent rounds approximately 50 nM, a 1/10 dilution of an optimizedPCR reaction, using conditions described herein, is used). The preferredDNA transcription templates are described below (where ARC254 and ARC256transcribe under all 2′-OMe conditions and ARC255 transcribes under rRmYconditions).

SEQ ID NO: 1 5′-CATCGATGCTAGTCGTAACGATCCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCGAGAACGTTCTCTCCTCTCCCTATAGTGAGTCGTATTA-3′ SEQ ID NO: 25′-CATGCATCGCGACTGACTAGCCGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGTAGAACGTTCTCTCCTCTCCCTATAGTGAGTCGTATTA-3′ SEQ ID NO: 35′-CATCGATCGATCGATCGACAGCGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGTAGAACGTTCTCTCCTCTCCCTATAGTGAGTCGTATTA-3′

Under rN transcription conditions of the present invention, thetranscription reaction mixture comprises 2′-OH adenosine triphosphates(ATP), 2′-OH guanosine triphosphates (GTP), 2′-OH cytidine triphosphates(CTP), and 2′-OH uridine triphosphates (UTP). The modifiedoligonucleotides produced using the rN transcription mixtures of thepresent invention comprise substantially all 2′-OH adenosine, 2′-OHguanosine, 2′-OH cytidine, and 2′-OH uridine. In a preferred embodimentof rN transcription, the resulting modified oligonucleotides comprise asequence where at least 80% of all adenosine nucleotides are 2′-OHadenosine, at least 80% of all guanosine nucleotides are 2′-OHguanosine, at least 80% of all cytidine nucleotides are 2′-OH cytidine,and at least 80% of all uridine nucleotides are 2′-OH uridine. In a morepreferred embodiment of rN transcription, the resulting modifiedoligonucleotides of the present invention comprise a sequence where atleast 90% of all adenosine nucleotides are 2′-OH adenosine, at least 90%of all guanosine nucleotides are 2′-OH guanosine, at least 90% of allcytidine nucleotides are 2′-OH cytidine, and at least 90% of all uridinenucleotides are 2′-OH uridine. In a most preferred embodiment of rNtranscription, the modified oligonucleotides of the present inventioncomprise a sequence where 100% of all adenosine nucleotides are 2′-OHadenosine, 100% of all guanosine nucleotides are 2′-OH guanosine, 100%of all cytidine nucleotides are 2′-OH cytidine, and 100% of all uridinenucleotides are 2′-OH uridine.

Under rRmY transcription conditions of the present invention, thetranscription reaction mixture comprises 2′-OH adenosine triphosphates,2′-OH guanosine triphosphates, 2′-OMe cytidine triphosphates, and 2′-OMeuridine triphosphates. The modified oligonucleotides produced using therRmY transcription mixtures of the present invention comprisesubstantially all 2′-OH adenosine, 2′-OH guanosine, 2′-OMe cytidine and2′-OMe uridine. In a preferred embodiment, the resulting modifiedoligonucleotides comprise a sequence where at least 80% of all adenosinenucleotides are 2′-OH adenosine, at least 80% of all guanosinenucleotides are 2′-OH guanosine, at least 80% of all cytidinenucleotides are 2′-OMe cytidine and at least 80% of all uridinenucleotides are 2′-OMe uridine. In a more preferred embodiment, theresulting modified oligonucleotides comprise a sequence where at least90% of all adenosine nucleotides are 2′-OH adenosine, at least 90% ofall guanosine nucleotides are 2′-OH guanosine, at least 90% of allcytidine nucleotides are 2′-OMe cytidine and at least 90% of all uridinenucleotides are 2′-OMe uridine In a most preferred embodiment, theresulting modified oligonucleotides comprise a sequence where 100% ofall adenosine nucleotides are 2′-OH adenosine, 100% of all guanosinenucleotides are 2′-OH guanosine, 100% of all cytidine nucleotides are2′-OMe cytidine and 100% of all uridine nucleotides are 2′-OMe uridine.

Under dRmY transcription conditions of the present invention, thetranscription reaction mixture comprises 2′-deoxy adenosinetriphosphates, 2′-deoxy guanosine triphosphates, 2′-O-methyl cytidinetriphosphates, and 2′-O-methyl uridine triphosphates. The modifiedoligonucleotides produced using the dRmY transcription conditions of thepresent invention comprise substantially all 2′-deoxy adenosine,2′-deoxy guanosine, 2′-O-methyl cytidine, and 2′-O-methyl uridine. In apreferred embodiment, the resulting modified oligonucleotides of thepresent invention comprise a sequence where at least 80% of alladenosine nucleotides are 2′-deoxy adenosine, at least 80% of allguanosine nucleotides are 2′-deoxy guanosine, at least 80% of allcytidine nucleotides are 2′-O-methyl cytidine, and at least 80% of alluridine nucleotides are 2′-O-methyl uridine. In a more preferredembodiment, the resulting modified oligonucleotides of the presentinvention comprise a sequence where at least 90% of all adenosinenucleotides are 2′-deoxy adenosine, at least 90% of all guanosinenucleotides are 2′-deoxy guanosine, at least 90% of all cytidinenucleotides are 2′-O-methyl cytidine, and at least 90% of all uridinenucleotides are 2′-O-methyl uridine. In a most preferred embodiment, theresulting modified oligonucleotides of the present invention comprise asequence where 100% of all adenosine nucleotides are 2′-deoxy adenosine,100% of all guanosine nucleotides are 2′-deoxy guanosine, 100% of allcytidine nucleotides are 2′-O-methyl cytidine, and 100% of all uridinenucleotides are 2′-O-methyl uridine.

Under rGmH transcription conditions of the present invention, thetranscription reaction mixture comprises 2′-OH guanosine triphosphates,2′-O-methyl cytidine triphosphates, 2′-O-methyl uridine triphosphates,and 2′-O-methyl adenosine triphosphates. The modified oligonucleotidesproduced using the rGmH transcription mixtures of the present inventioncomprise substantially all 2′-OH guanosine, 2′-O-methyl cytidine,2′-O-methyl uridine, and 2′-O-methyl adenosine. In a preferredembodiment, the resulting modified oligonucleotides comprise a sequencewhere at least 80% of all guanosine nucleotides are 2′-OH guanosine, atleast 80% of all cytidine nucleotides are 2′-O-methyl cytidine, at least80% of all uridine nucleotides are 2′-O-methyl uridine, and at least 80%of all adenosine nucleotides are 2′-O-methyl adenosine. In a morepreferred embodiment, the resulting modified oligonucleotides comprise asequence where at least 90% of all guanosine nucleotides are 2′-OHguanosine, at least 90% of all cytidine nucleotides are 2′-O-methylcytidine, at least 90% of all uridine nucleotides are 2′-O-methyluridine, and at least 90% of all adenosine nucleotides are 2′-O-methyladenosine. In a most preferred embodiment, the resulting modifiedoligonucleotides comprise a sequence where 100% of all guanosinenucleotides are 2′-OH guanosine, 100% of all cytidine nucleotides are2′-O-methyl cytidine, 100% of all uridine nucleotides are 2′-O-methyluridine, and 100% of all adenosine nucleotides are 2′-O-methyladenosine.

Under r/mGmH transcription conditions of the present invention, thetranscription reaction mixture comprises 2′-O-methyl adenosinetriphosphate, 2′-O-methyl cytidine triphosphate, 2′-O-methyl guanosinetriphosphate, 2′-O-methyl uridine triphosphate and 2′-OH guanosinetriphosphate. The resulting modified oligonucleotides produced using ther/mGmH transcription mixtures of the present invention comprisesubstantially all 2′-O-methyl adenosine, 2′-O-methyl cytidine,2′-O-methyl guanosine, and 2′-O-methyl uridine, wherein the populationof guanosine nucleotides has a maximum of about 10% 2′-OH guanosine. Ina preferred embodiment, the resulting r/mGmH modified oligonucleotidesof the present invention comprise a sequence where at least 80% of alladenosine nucleotides are 2′-O-methyl adenosine, at least 80% of allcytidine nucleotides are 2′-O-methyl cytidine, at least 80% of allguanosine nucleotides are 2′-O-methyl guanosine, at least 80% of alluridine nucleotides are 2′-O-methyl uridine, and no more than about 10%of all guanosine nucleotides are 2′-OH guanosine. In a more preferredembodiment, the resulting modified oligonucleotides comprise a sequencewhere at least 90% of all adenosine nucleotides are 2′-O-methyladenosine, at least 90% of all cytidine nucleotides are 2′-O-methylcytidine, at least 90% of all guanosine nucleotides are 2′-O-methylguanosine, at least 90% of all uridine nucleotides are 2′-O-methyluridine, and no more than about 10% of all guanosine nucleotides are2′-OH guanosine. In a most preferred embodiment, the resulting modifiedoligonucleotides comprise a sequence where 100% of all adenosinenucleotides are 2′-O-methyl adenosine, 100% of all cytidine nucleotidesare 2′-O-methyl cytidine, 90% of all guanosine nucleotides are2′-O-methyl guanosine, and 100% of all uridine nucleotides are2′-O-methyl uridine, and no more than about 10% of all guanosinenucleotides are 2′-OH guanosine.

Under mRmY transcription conditions of the present invention, thetranscription mixture comprises only 2′-O-methyl adenosine triphosphate,2′-O-methyl cytidine triphosphate, 2′-O-methyl guanosine triphosphate,2′-O-methyl uridine triphosphate. The resulting modifiedoligonucleotides produced using the mRmY transcription mixture of thepresent invention comprise a sequence where 100% of all adenosinenucleotides are 2′-O-methyl adenosine, 100% of all cytidine nucleotidesare 2′-O-methyl cytidine, 100% of all guanosine nucleotides are2′-O-methyl guanosine, and 100% of all uridine nucleotides are2′-O-methyl uridine.

Under fGmH transcription conditions of the present invention, thetranscription reaction mixture comprises 2′-O-methyl adenosinetriphosphates, 2′-O-methyl uridine triphosphates, 2′-O-methyl cytidinetriphosphates, and 2′-F guanosine triphosphates. The modifiedoligonucleotides produced using the fGmH transcription conditions of thepresent invention comprise substantially all 2′-O-methyl adenosine,2′-O-methyl uridine, 2′-O-methyl cytidine, and 2′-F guanosine. In apreferred embodiment, the resulting modified oligonucleotides comprise asequence where at least 80% of all adenosine nucleotides are 2′-O-methyladenosine, at least 80% of all uridine nucleotides are 2′-O-methyluridine, at least 80% of all cytidine nucleotides are 2′-O-methylcytidine, and at least 80% of all guanosine nucleotides are 2′-Fguanosine. In a more preferred embodiment, the resulting modifiedoligonucleotides comprise a sequence where at least 90% of all adenosinenucleotides are 2′-O-methyl adenosine, at least 90% of all uridinenucleotides are 2′-O-methyl uridine, at least 90% of all cytidinenucleotides are 2′-O-methyl cytidine, and at least 90% of all guanosinenucleotides are 2′-F guanosine. In a most preferred embodiment, theresulting modified oligonucleotides comprise a sequence where 100% ofall adenosine nucleotides are 2′-O-methyl adenosine, 100% of all uridinenucleotides are 2′-O-methyl uridine, 100% of all cytidine nucleotidesare 2′-O-methyl cytidine, and 100% of all guanosine nucleotides are 2′-Fguanosine.

Under dAmB transcription conditions of the present invention, thetranscription reaction mixture comprises 2′-deoxy adenosinetriphosphates, 2′-O-methyl cytidine triphosphates, 2′-O-methyl guanosinetriphosphates, and 2′-O-methyl uridine triphosphates. The modifiedoligonucleotides produced using the dAmB transcription mixtures of thepresent invention comprise substantially all 2′-deoxy adenosine,2′-O-methyl cytidine, 2′-O-methyl guanosine, and 2′-O-methyl uridine. Ina preferred embodiment, the resulting modified oligonucleotides comprisea sequence where at least 80% of all adenosine nucleotides are 2′-deoxyadenosine, at least 80% of all cytidine nucleotides are 2′-O-methylcytidine, at least 80% of all guanosine nucleotides are 2′-O-methylguanosine, and at least 80% of all uridine nucleotides are 2′-O-methyluridine. In a more preferred embodiment, the resulting modifiedoligonucleotides comprise a sequence where at least 90% of all adenosinenucleotides are 2′-deoxy adenosine, at least 90% of all cytidinenucleotides are 2′-O-methyl cytidine, at least 90% of all guanosinenucleotides are 2′-O-methyl guanosine, and at least 90% of all uridinenucleotides are 2′-O-methyl uridine. In a most preferred embodiment, theresulting modified oligonucleotides of the present invention comprise asequence where 100% of all adenosine nucleotides are 2′-deoxy adenosine,100% of all cytidine nucleotides are 2′-O-methyl cytidine, 100% of allguanosine nucleotides are 2′-O-methyl guanosine, and 100% of all uridinenucleotides are 2′-O-methyl uridine.

In each case, the transcription products can then be used for input intothe SELEX™ process to identify aptamers and/or to determine a conservedsequences that has binding specificity to a given target. The resultingsequences are already partially stabilized, eliminating this step fromthe post-SELEX™ process to arrive at an optimized aptamer sequence andgiving a more highly stabilized aptamer as a result. Another advantageof the 2′-OMe SELEX™ process is that the resulting sequences are likelyto have fewer 2′-OH nucleotides required in the sequence, possibly none.To the extent 2′OH nucleotides remain they may be removed by performingpost-SELEX™ modifications.

As described below, lower but still useful yields of transcripts fullyincorporating 2′ substituted nucleotides can be obtained underconditions other than the optimized conditions described above. Forexample, variations to the above transcription conditions include:

The HEPES buffer concentration can range from 0 to 1 M. The presentinvention also contemplates the use of other buffering agents having apKa between 5 and 10 including, for example,Tris-hydroxymethyl-aminomethane.

The DTT concentration can range from 0 to 400 mM. The methods of thepresent invention also provide for the use of other reducing agentsincluding, for example, mercaptoethanol.

The spermidine and/or spermidine concentration can range from 0 to 20mM.

The PEG-8000 concentration can range from 0 to 50% (w/v). The methods ofthe present invention also provide for the use of other hydrophilicpolymer including, for example, other molecular weight PEG or otherpolyalkylene glycols.

The Triton X-100 concentration can range from 0 to 0.1% (w/v). Themethods of the present invention also provide for the use of othernon-ionic detergents including, for example, other detergents, includingother Triton-X detergents.

The MgCl₂ concentration can range from 0.5 mM to 50 mM. The MnCl₂concentration can range from 0.15 mM to 15 mM. Both MgCl₂ and MnCl₂ mustbe present within the ranges described and in a preferred embodiment arepresent in about a 10 to about 3 ratio of MgCl₂:MnCl₂, preferably, theratio is about 3-5:1, more preferably, the ratio is about 3-4:1.

The 2′-OMe NTP concentration (each NTP) can range from 5 μM to 5 mM.

The 2′-OH GTP concentration can range from 0 μM to 300 μM.

The 2′-OH GMP concentration can range from 0 to 5 mM.

The pH can range from pH 6 to pH 9. The methods of the present inventioncan be practiced within the pH range of activity of most polymerasesthat incorporate modified nucleotides. In addition, the methods of thepresent invention provide for the optional use of chelating agents inthe transcription reaction condition including, for example, EDTA, EGTA,and DTT.

Aptamer Medicinal Chemistry

Aptamer Medicinal Chemistry is an aptamer improvement technique in whichsets of variant aptamers are chemically synthesized. These sets ofvariants typically differ from the parent aptamer by the introduction ofa single substituent, and differ from each other by the location of thissubstituent. These variants are then compared to each other and to theparent. Improvements in characteristics may be profound enough that theinclusion of a single substituent may be all that is necessary toachieve a particular therapeutic criterion.

Alternatively the information gleaned from the set of single variantsmay be used to design further sets of variants in which more than onesubstituent is introduced simultaneously. In one design strategy, all ofthe single substituent variants are ranked, the top 4 are chosen and allpossible double (6), triple (4) and quadruple (1) combinations of these4 single substituent variants are synthesized and assayed. In a seconddesign strategy, the best single substituent variant is considered to bethe new parent and all possible double substituent variants that includethis highest-ranked single substituent variant are synthesized andassayed. Other strategies may be used, and these strategies may beapplied repeatedly such that the number of substituents is graduallyincreased while continuing to identify further-improved variants.

Aptamer Medicinal Chemistry may be used particularly as a method toexplore the local, rather than the global, introduction of substituents.Because aptamers are discovered within libraries that are generated bytranscription, any substituents that are introduced during the SELEX™process must be introduced globally. For example, if it is desired tointroduce phosphorothioate linkages between nucleotides then they canonly be introduced at every A (or every G, C, T, U etc.) (globallysubstituted). Aptamers which require phosphorothioates at some As (orsome G, C, T, U etc.) (locally substituted) but cannot tolerate it atother As cannot be readily discovered by this process.

The kinds of substituent that can be utilized by the Aptamer MedicinalChemistry process are only limited by the ability to generate them assolid-phase synthesis reagents and introduce them into an oligomersynthesis scheme. The process is certainly not limited to nucleotidesalone. Aptamer Medicinal Chemistry schemes may include substituents thatintroduce steric bulk, hydrophobicity, hydrophilicity, lipophilicity,lipophobicity, positive charge, negative charge, neutral charge,zwitterions, polarizability, nuclease-resistance, conformationalrigidity, conformational flexibility, protein-binding characteristics,mass etc. Aptamer Medicinal Chemistry schemes may includebase-modifications, sugar-modifications or phosphodiesterlinkage-modifications.

When considering the kinds of substituents that are likely to bebeneficial within the context of a therapeutic aptamer, it may bedesirable to introduce substitutions that fall into one or more of thefollowing categories:

-   -   (1) Substituents already present in the body, e.g., 2′-deoxy,        2′-ribo, 2′-O-methyl purines or pyrimidines or 5-methyl        cytosine.    -   (2) Substituents already part of an approved therapeutic, e.g.,        phosphorothioate-linked oligonucleotides.    -   (3) Substituents that hydrolyze or degrade to one of the above        two categories, e.g., methylphosphonate-linked oligonucleotides.

The vWF aptamers of the invention include aptamers developed throughaptamer medicinal chemistry as described herein.

Von Willebrand Factor Specific Binding Aptamers

The materials of the present invention comprise a series of nucleic acidaptamers of 29 to 76 nucleotides in length which bind specifically tovon Willebrand Factor. In one embodiment, materials of the presentinvention comprise a series of nucleic acid aptamers of 29 to 76nucleotides in length which bind specifically to von Willebrand Factorand which functionally modulate, e.g., block, an activity of vonWillebrand Factor in vivo and/or cell-based assays.

Aptamers specifically capable of binding and modulating full length vonWillebrand Factor and/or von Willebrand Factor domain A1 are set forthherein. These aptamers provide a low-toxicity, safe, and effectivemodality of treating and/or preventing cardiovascular diseases ordisorders. In one embodiment, the aptamers of the invention are used ina method to treat and/or prevent coronary artery diseases, including anyone of the disorders selected from the group consisting of: arterialthrombosis and acute coronary syndromes such as unstable angina andmyocardial infarction which are known to be caused by or otherwiseassociated with von Willebrand Factor mediated platelet aggregation. Inparticular embodiments, the aptamers of the invention are used in amethod to treat and/or prevent coronary artery diseases, including anyone of the disorders selected from the group consisting of: arterialthrombosis and acute coronary syndromes such as unstable angina andmyocardial infarction which are known to be caused by or otherwiseassociated with von Willebrand Factor mediated platelet aggregationwhile minimizing bleeding side effects. In another embodiment, theaptamers of the invention are used in a method to treat and/or preventperipheral vascular diseases which are known to be caused by orotherwise associated with von Willebrand Factor mediated plateletaggregation. In a particular embodiment, the aptamers of the inventionare used in a method to treat and/or prevent peripheral vasculardiseases which are known to be caused by or otherwise associated withvon Willebrand Factor mediated platelet aggregation, preferably, whileminimizing bleeding side effects. In another embodiment, the aptamers ofthe invention are used to treat and/or prevent cerebrovascular diseases,including any one of the disorders selected from the group consistingof: transient cerebral ischemic attack, stroke and carotid stenosiswhich are known to be caused by or otherwise associated with vonWillebrand Factor mediated platelet aggregation, preferably, whileminimizing bleeding side effects. Further, aptamers of the invention areuseful to inhibit von Willebrand Factor mediated platelet aggregation ina subject prior to, during, and/or after a subject has undergonepercutaneous coronary intervention including angioplasty, thrombolytictreatment or coronary bypass surgery. Aptamers of the invention are alsouseful for maintaining blood vessel patency in a subject prior to,during and/or after the subject has undergone coronary bypass surgery.The aptamers of the invention are also useful for treating a patientundergoing dialysis. The aptamers of the invention are also useful forinhibiting von Willebrand Factor mediated thrombosis in a subject,preferably while also minimizing bleeding side effects. The thrombosisto be treated and/or inhibited may be associated with an inflammatoryresponse.

In one embodiment, the von Willebrand Factor specific binding aptamerfor use in therapeutics and/or diagnostics is selected from the groupconsisting of: SEQ ID NOS 11 to 50, SEQ ID NOS 54 to 94, SEQ ID NOS 98to 164, SEQ ID NO 165, SEQ ID NO 169, SEQ ID NO 172, SEQ ID NO 174, SEQID NO 177, SEQ ID NO 180, SEQ ID NO 183, SEQ ID NO 186, SEQ ID NO 189,SEQ ID NO 192, SEQ ID NO 198, SEQ ID NO 201, SEQ ID NO 205, SEQ ID NO208, SEQ ID NOS 212-214, ARC1115 (SEQ ID NO 221), ARC1172 (SEQ ID NO222), ARC1194 (SEQ ID NO 223) to ARC1240 (SEQ ID NO 269), ARC1338 (SEQID NO 273) to ARC1346 (SEQ ID NO 281), ARC1361 (SEQ ID NO 284) toARC1381 (SEQ ID NO 304), ARC1524 (SEQ ID NO 305), ARC1526 (SEQ ID NO307) to ARC1535 (SEQ ID NO 316), ARC1546 (SEQ ID NO 317), ARC1635,ARC1759 (SEQ ID NO 318), ARC1779 (SEQ ID NO 320) to ARC1780 and ARC1884(SEQ ID NO 322) to ARC1885 (SEQ ID NO 323).

In another embodiment, von Willebrand Factor specific binding aptamersfor use as therapeutics and/or diagnostics include any one of thefollowing sequences: SEQ ID NO 23, SEQ ID NO 44, SEQ ID NO 49, SEQ IDNOS 98-100, SEQ ID NO 106, SEQ ID NO 109, SEQ ID NOS 114 to 115, SEQ IDNO 118, SEQ ID NO 127, SEQ ID NO 134, SEQ ID NO 164, SEQ ID NO165, SEQID NO 169, SEQ ID NO 172, SEQ ID NO 174, SEQ ID NO 177, SEQ ID NO 180,SEQ ID NO 183, SEQ ID NO 186, SEQ ID NO 189, SEQ ID NO 192, SEQ ID NO198, SEQ ID NO 201, SEQ ID NO 208, and SEQ ID NOS 212 to 214. In someembodiments, von Willebrand Factor specific bind aptamers for use astherapeutics and/or diagnostics include any one of the followingsequences: ARC1029 (SEQ ID NO 214), ARC1115 (SEQ ID NO 221), ARC1172(SEQ ID NO 222), ARC1346 (SEQ ID NO 281), ARC1361 (SEQ ID NO 284),ARC1368 (SEQ ID NO 291), ARC1635 (SEQ ID NO 319), ARC1759 (SEQ ID NO318), ARC1779 (SEQ ID NO 320), ARC 1780 (SEQ ID NO 321), ARC1884 (SEQ IDNO 322) to ARC1885 (SEQ ID NO 323).

Other aptamers of the invention that bind von Willebrand Factor aredescribed below in Examples 1 and 2.

These aptamers may include modifications as described herein including,e.g., conjugation to lipophilic or high molecular weight compounds(e.g., PEG), incorporation of a capping moiety, incorporation ofmodified nucleotides, and phosphate back bone modification (includingincorporation of phosphorothioate into the phosphate backbone).

In one embodiment of the invention an isolated, non-naturally occurringaptamer that binds to von Willebrand Factor is provided. In anotherembodiment, the aptamer of the invention modulates a function of vonWillebrand Factor. In another embodiment, the aptamer of the inventioninhibits a function of von Willebrand Factor while in another embodimentthe aptamer stimulates a function of von Willebrand Factor. In anotherembodiment of the invention, the aptamer binds and/or modulates afunction of a von Willebrand Factor variant. A von Willebrand Factorvariant as used herein encompasses variants that perform essentially thesame function as a von Willebrand Factor function, preferably comprisessubstantially the same structure and in some embodiments comprises atleast 70% sequence identity, preferably at least 80% sequence identity,more preferably at least 90% sequence identity, and more preferably atleast 95% sequence identity to the amino acid sequence of human vonWillebrand Factor.

In another embodiment of the invention, the aptamer has substantiallythe same ability to bind von Willebrand Factor as that of an aptamercomprising any one of SEQ ID NOS 11 to 50, SEQ ID NOS 54 to 94, SEQ IDNOS 98 to 164, SEQ ID NO 165, SEQ ID NO 169, SEQ ID NO 172, SEQ ID NO174, SEQ ID NO 177, SEQ ID NO 180, SEQ ID NO 183, SEQ ID NO 186, SEQ IDNO 189, SEQ ID NO 192, SEQ ID NO 198, SEQ ID NO 201, SEQ ID NO 205,SEQID NO 208,SEQ ID NOS 212-214,ARC1115,ARC1172(SEQ ID NO 222)(SEQ ID NO222), ARC1194 (SEQ ID NO 223) to ARC1240 (SEQ ID NO 269), ARC1338 (SEQID NO 273) to ARC1346 (SEQ ID NO 281), ARC1361 (SEQ ID NO 284) toARC1381 (SEQ ID NO 304), ARC1524 (SEQ ID NO 305), ARC1526 (SEQ ID NO307) to ARC1535 (SEQ ID NO 316), ARC1546 (SEQ ID NO 317), ARC1635,ARC1759 (SEQ ID NO 318), ARC1779 (SEQ ID NO 320) to ARC1780 (SEQ ID NO321) and ARC1884 (SEQ ID NO 322) to ARC1885 (SEQ ID NO 323). In anotherembodiment of the invention, the aptamer has substantially the samestructure and ability to bind von Willebrand Factor as that of anaptamer comprising any one of SEQ ID NOS 11 to 50, SEQ ID NOS 54 to 94,SEQ ID NOS 98 to 165, SEQ ID NO 169, SEQ ID NO 172, SEQ ID NO 174, SEQID NO 177, SEQ ID NO 180, SEQ ID NO 183, SEQ ID NO 186, SEQ ID NO 189,SEQ ID NO 192, SEQ ID NO 198, SEQ ID NO 201, SEQ ID NO 205, SEQ ID NO208, SEQ ID NOS 212-214, ARC1115, ARC1172 (SEQ ID NO 222) (SEQ ID NO222), ARC1194 (SEQ ID NO 223) to ARC1240 (SEQ ID NO 269), ARC1338 (SEQID NO 273) to ARC1346 (SEQ ID NO 281), ARC1361 (SEQ ID NO 284) toARC1381 (SEQ ID NO 304), ARC1524 (SEQ ID NO 305), ARC1526 (SEQ ID NO307) to ARC1535 (SEQ ID NO 316), ARC1546 (SEQ ID NO 317), ARC1635,ARC1759 (SEQ ID NO 318), ARC1779 (SEQ ID NO 320) to ARC1780 (SEQ ID NO321) and ARC1884 (SEQ ID NO 322) to ARC1885 (SEQ ID NO 323). In anotherembodiment, the aptamers of the invention comprise a sequence accordingto any one of SEQ ID NOS 11 to 50, SEQ ID NOS 54 to 94, SEQ ID NOS 98 to165, SEQ ID NO 169, SEQ ID NO 172, SEQ ID NO 174, SEQ ID NO 177, SEQ IDNO 180, SEQ ID NO 183, SEQ ID NO 186, SEQ ID NO 189, SEQ ID NO 192, SEQID NO 198, SEQ ID NO 201, SEQ ID NO 205, SEQ ID NO 208, SEQ ID NOS212-213, ARC1115, ARC1172 (SEQ ID NO 222) (SEQ ID NO 222), ARC1194 (SEQID NO 223) to ARC1240 (SEQ ID NO 269), ARC1338 (SEQ ID NO 273) toARC1346 (SEQ ID NO 281), ARC1361 (SEQ ID NO 284) to ARC1381 (SEQ ID NO304), ARC1524 (SEQ ID NO 305), ARC1526 (SEQ ID NO 307) to ARC1535 (SEQID NO 316), ARC1546 (SEQ ID NO 317), ARC1635, ARC1759 (SEQ ID NO 318),ARC1779 (SEQ ID NO 320) to ARC1780 (SEQ ID NO 321) and ARC1884 (SEQ IDNO 322) to ARC1885 (SEQ ID NO 323). In another embodiment, the aptamersof the invention comprise a sequence that is at least 80% identical,preferably at least 90% identical and in some embodiments at least 95%identical to a sequence according to any one of SEQ ID NOS 11 to 50, SEQID NOS 54 to 94, SEQ ID NOS 98 to 164, SEQ ID NO 165, SEQ ID NO 169, SEQID NO 172, SEQ ID NO 174, SEQ ID NO 177, SEQ ID NO 180, SEQ ID NO 183,SEQ ID NO 186, SEQ ID NO 189, SEQ ID NO 192, SEQ ID NO 198, SEQ ID NO201, SEQ ID NO 205, SEQ ID NO 208, SEQ ID NOS 212-214, ARC 115, ARC1172(SEQ ID NO 222) (SEQ ID NO 222), ARC1194 (SEQ ID NO 223) to ARC1240 (SEQID NO 269), ARC1338 (SEQ ID NO 273) to ARC1346 (SEQ ID NO 281), ARC1361(SEQ ID NO 284) to ARC1381 (SEQ ID NO 304), ARC1524 (SEQ ID NO 305),ARC1526 (SEQ ID NO 307) to ARC1535 (SEQ ID NO 316), ARC1546 (SEQ ID NO317), ARC1635, ARC1759 (SEQ ID NO 318), ARC1779 (SEQ ID NO 320) toARC1780 (SEQ ID NO 321) and ARC1884 (SEQ ID NO 322) to ARC1885 (SEQ IDNO 323). In another embodiment, the aptamers of the inventionspecifically bind von Willebrand Factor and comprise a sequence of 30contiguous nucleotides that are identical to 30 contiguous nucleotidesin any one of the aptamers selected from the group consisting of: SEQ IDNOS 11 to 50, SEQ ID NOS 54 to 94, SEQ ID NOS 98 to 164, SEQ ID NO 165,SEQ ID NO 169, SEQ ID NO 172, SEQ ID NO 174, SEQ ID NO 177, SEQ ID NO180, SEQ ID NO 183, SEQ ID NO 186, SEQ ID NO 189, SEQ ID NO 192, SEQ IDNO 198, SEQ ID NO 201, SEQ ID NO 205, SEQ ID NO 208, SEQ ID NOS 212-214,ARC1115, ARC1172 (SEQ ID NO 222) (SEQ ID NO 222), ARC1194 (SEQ ID NO223) to ARC1240 (SEQ ID NO 269), ARC1338 (SEQ ID NO 273) to ARC1346 (SEQID NO 281), ARC1361 (SEQ ID NO 284) to ARC1381 (SEQ ID NO 304), ARC1524(SEQ ID NO 305), ARC1526 (SEQ ID NO 307) to ARC1535 (SEQ ID NO 316),ARC1546 (SEQ ID NO 317), ARC1635, ARC1759 (SEQ ID NO 318), ARC1779 (SEQID NO 320) to ARC1780 (SEQ ID NO 321) and ARC1884 (SEQ ID NO 322) toARC1885 (SEQ ID NO 323). In another embodiment, the aptamers of theinvention are used as an active ingredient in pharmaceuticalcompositions. In another embodiment, the aptamers of the invention orcompositions comprising the aptamers of the invention are used to treatthrombotic disease such as cardiovascular disorders, including acutecoronary syndrome; peripheral arterial disease; and cerebrovasculardisorders, including stroke. In some embodiments, the aptamers of theinvention or compositions comprising the aptamers of the invention areuse to treat, prevent or ameliorate a disorder selected from the groupconsisting of: essential thrombocytopenia,: thrombotic thrombocopenicpurpura (“TTP”), Type IIb von Willebrand's disease, pseudo vonWillebrand disease, peripheral artery disease, e.g. peripheral arterialocclusive disease, unstable angina, angina pectoris, arterialthrombosis, atherosclerosis, myocardial infarction, acute coronarysyndrome, atrial fibrillation, carotid stenosis, cerebral infarction,cerebral thrombosis, ischemic stroke, and transient cerebral ischemicattack. In some embodiments, the pharmaceutical composition of theinvention is administered prior to/during and/or after dialysis, CABGsurgery, percutaneous coronary intervention or heart valve replacement.

In some embodiments, aptamer therapeutics of the present invention havegreat affinity and specificity to their targets while reducing thedeleterious side effects from non-naturally occurring nucleotidesubstitutions if the aptamer therapeutics break down in the body ofpatients or subjects. In some embodiments, the therapeutic compositionscontaining the aptamer therapeutics of the present invention are free ofor have a reduced amount of fluorinated nucleotides.

The aptamers of the present invention can be synthesized using anyoligonucleotide synthesis techniques known in the art including solidphase oligonucleotide synthesis techniques (see, e.g., Froehler et al.,Nucl. Acid Res. 14:5399-5467 (1986) and Froehler et al., Tet. Lett.27:5575-5578 (1986)) and solution phase methods such as triestersynthesis methods (see, e.g., Sood et al., Nucl. Acid Res. 4:2557 (1977)and Hirose et al., Tet. Lett, 28:2449 (1978)) both of which are wellknown in the art.

Pharmaceutical Compositions

The invention also includes pharmaceutical compositions containingaptamer molecules that bind to von Willebrand Factor. In someembodiments, the compositions are suitable for internal use and includean effective amount of a pharmacologically active compound of theinvention, alone or in combination, with one or more pharmaceuticallyacceptable carriers. The compounds are especially useful in that theyhave very low, if any toxicity.

Compositions of the invention can be used to treat or prevent apathology, such as a disease or disorder, or alleviate the symptoms ofsuch disease or disorder in a patient. For example, compositions of thepresent invention can be used to treat or prevent a pathology associatedwith platelet aggregation. In some embodiments, the disease to betreated, prevented or ameliorated is selected from the group consistingof: essential thrombocytopenia,: thrombotic thrombocopenic purpura(“TTP”), Type IIb von Willebrand's disease, pseudo von Willebranddisease, peripheral artery disease, e.g. peripheral arterial occlusivedisease, unstable angina, angina pectoris, arterial thrombosis,atherosclerosis, myocardial infarction, acute coronary syndrome, atrialfibrillation, carotid stenosis, cerebral infarction, cerebralthrombosis, ischemic stroke, and transient cerebral ischemic attack. Insome embodiments, the pharmaceutical composition of the invention isadministered prior to, during and/or after dialysis, CABG surgery,percutaneous coronary intervention or heart valve replacement.

Compositions of the invention are useful for administration to a subjectsuffering from, or predisposed to, a disease or disorder which isrelated to or derived from a target to which the aptamers of theinvention specifically bind.

Compositions of the invention can be used in a method for treating apatient or subject having a pathology. The method involves administeringto the patient or subject an aptamer or a composition comprisingaptamers that bind to von Willebrand Factor involved with the pathology,so that binding of the aptamer to the target alters the biologicalfunction of von Willebrand Factor, thereby treating the pathology.

The patient or subject having a pathology, i.e., the patient or subjecttreated by the methods of this invention can be a mammal, moreparticularly a vertebrate, or more particularly, a human.

In practice, the aptamers or their pharmaceutically acceptable salts,are administered in amounts which will be sufficient to exert theirdesired biological activity, e.g., preventing vWF dependent plateletaggregation.

One aspect of the invention comprises an aptamer composition of theinvention in combination with other treatments for thrombotic relateddisorders. The aptamer composition of the invention may contain, forexample, more than one aptamer, e.g. an anti-thrombin aptamer and ananti-vWF aptamer. In some examples, an aptamer composition of theinvention, containing one or more aptamers of the invention, isadministered in combination with another useful composition such as ananti-inflammatory agent, an immunosuppressant, an antiviral agent, orthe like. In general, the currently available dosage forms of the knowntherapeutic agents for use in such combinations will be suitable.

“Combination therapy” (or “co-therapy”) includes the administration ofan aptamer composition of the invention and at least a second agent aspart of a specific treatment regimen intended to provide the beneficialeffect from the co-action of these therapeutic agents. The beneficialeffect of the combination includes, but is not limited to,pharmacokinetic or pharmacodynamic co-action resulting from thecombination of therapeutic agents. Administration of these therapeuticagents in combination typically is carried out over a defined timeperiod (usually minutes, hours, days or weeks depending upon thecombination selected).

“Combination therapy” may, but generally is not, intended to encompassthe administration of two or more of these therapeutic agents as part ofseparate monotherapy regimens that incidentally and arbitrarily resultin the combinations of the present invention. “Combination therapy” isintended to embrace administration of these therapeutic agents in asequential manner, that is, wherein each therapeutic agent isadministered at a different time, as well as administration of thesetherapeutic agents, or at least two of the therapeutic agents, in asubstantially simultaneous manner. Substantially simultaneousadministration can be accomplished, for example, by administering to thesubject a single capsule having a fixed ratio of each therapeutic agentor in multiple, single capsules for each of the therapeutic agents.

Sequential or substantially simultaneous administration of eachtherapeutic agent can be effected by any appropriate route including,but not limited to, topical routes, oral routes, intravenous routes,intramuscular routes, and direct absorption through mucous membranetissues. The therapeutic agents can be administered by the same route orby different routes. For example, a first therapeutic agent of thecombination selected may be administered by injection while the othertherapeutic agents of the combination may be administered topically.

Alternatively, for example, all therapeutic agents may be administeredtopically or all therapeutic agents may be administered by injection.The sequence in which the therapeutic agents are administered is notnarrowly critical unless noted otherwise. “Combination therapy” also canembrace the administration of the therapeutic agents as described abovein further combination with other biologically active ingredients. Wherethe combination therapy further comprises a non-drug treatment, thenon-drug treatment may be conducted at any suitable time so long as abeneficial effect from the co-action of the combination of thetherapeutic agents and non-drug treatment is achieved. For example, inappropriate cases, the beneficial effect is still achieved when thenon-drug treatment is temporally removed from the administration of thetherapeutic agents, perhaps by days or even weeks.

Therapeutic or pharmacological compositions of the present inventionwill generally comprise an effective amount of the active component(s)of the therapy, dissolved or dispersed in a pharmaceutically acceptablemedium. Pharmaceutically acceptable media or carriers include any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Supplementary active ingredients can also be incorporatedinto the therapeutic compositions of the present invention.

The preparation of pharmaceutical or pharmacological compositions willbe known to those of skill in the art in light of the presentdisclosure. Typically, such compositions may be prepared as injectables,either as liquid solutions or suspensions; solid forms suitable forsolution in, or suspension in, liquid prior to injection; as tablets orother solids for oral administration; as time release capsules; or inany other form currently used, including eye drops, creams, lotions,salves, inhalants and the like. The use of sterile formulations, such assaline-based washes, by surgeons, physicians or health care workers totreat a particular area in the operating field may also be particularlyuseful. Compositions may also be delivered via microdevice,microparticle or sponge.

Upon formulation, therapeutics will be administered in a mannercompatible with the dosage formulation, and in such amount as ispharmacologically effective. The formulations are easily administered ina variety of dosage forms. In a preferred embodiment the aptamer of theinvention is formulated as an injectable solution described above, butdrug release capsules and the like can also be employed.

In this context, the quantity of active ingredient and volume ofcomposition to be administered depends on the host animal to be treated.Precise amounts of active compound required for administration depend onthe judgment of the practitioner and are peculiar to each individual.

A minimal volume of a composition required to disperse the activecompounds is typically utilized. Suitable regimes for administration arealso variable, but would be typified by initially administering thecompound and monitoring the results and then giving further controlleddoses at further intervals. The effects of administration of theanti-vWF aptamer of the invention could be monitored by measuringplatelet aggregation formation such as measuring botrocetin inducedplatelet aggregation (“BIPA”) and/or shear force induced hemostatic plugformation using the PFA-100 instrument as described in Example 3 below.

For instance, for oral administration in the form of a tablet or capsule(e.g., a gelatin capsule), the active drug component can be combinedwith an oral, non-toxic pharmaceutically acceptable inert carrier suchas ethanol, glycerol, water and the like. Moreover, when desired ornecessary, suitable binders, lubricants, disintegrating agents andcoloring agents can also be incorporated into the mixture. Suitablebinders include starch, magnesium aluminum silicate, starch paste,gelatin, methylcellulose, sodium carboxymethylcellulose and/orpolyvinylpyrrolidone, natural sugars such as glucose or beta-lactose,corn sweeteners, natural and synthetic gums such as acacia, tragacanthor sodium alginate, polyethylene glycol, waxes and the like. Lubricantsused in these dosage forms include sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride,silica, talcum, stearic acid, its magnesium or calcium salt and/orpolyethyleneglycol and the like. Disintegrators include, withoutlimitation, starch, methyl cellulose, agar, bentonite, xanthan gumstarches, agar, alginic acid or its sodium salt, or effervescentmixtures, and the like. Diluents, include, e.g., lactose, dextrose,sucrose, mannitol, sorbitol, cellulose and/or glycine.

Injectable compositions are preferably aqueous isotonic solutions orsuspensions, and suppositories are advantageously prepared from fattyemulsions or suspensions. The compositions may be sterilized and/orcontain adjuvants, such as preserving, stabilizing, wetting oremulsifying agents, solution promoters, salts for regulating the osmoticpressure and/or buffers. In addition, they may also contain othertherapeutically valuable substances. The compositions are preparedaccording to conventional mixing, granulating or coating methods,respectively, and typically contain about 0.1 to 75%, preferably about 1to 50%, of the active ingredient.

The compounds of the invention can also be administered in such oraldosage forms as timed release and sustained release tablets or capsules,pills, powders, granules, elixirs, tinctures, suspensions, syrups andemulsions.

Liquid, particularly injectable compositions can, for example, beprepared by dissolving, dispersing, etc. The active compound isdissolved in or mixed with a pharmaceutically pure solvent such as, forexample, water, saline, aqueous dextrose, glycerol, ethanol, and thelike, to thereby form the injectable solution or suspension.Additionally, solid forms suitable for dissolving in liquid prior toinjection can be formulated.

The compounds of the present invention can be administered inintravenous (both bolus and infusion), intraperitoneal, subcutaneous orintramuscular form, all using forms well known to those of ordinaryskill in the pharmaceutical arts. Injectables can be prepared inconventional forms, either as liquid solutions or suspensions.

Parenteral injectable administration is generally used for subcutaneous,intramuscular or intravenous injections and infusions. Additionally, oneapproach for parenteral administration employs the implantation of aslow-release or sustained-released systems, which assures that aconstant level of dosage is maintained, according to U.S. Pat. No.3,710,795, incorporated herein by reference.

Furthermore, preferred compounds for the present invention can beadministered in intranasal form via topical use of suitable intranasalvehicles, inhalants, or via transdermal routes, using those forms oftransdermal skin patches well known to those of ordinary skill in thatart. To be administered in the form of a transdermal delivery system,the dosage administration will, of course, be continuous rather thanintermittent throughout the dosage regimen. Other preferred topicalpreparations include creams, ointments, lotions, aerosol sprays andgels, wherein the concentration of active ingredient would typicallyrange from 0.01% to 15%, w/w or w/v.

For solid compositions, excipients include pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum,cellulose, glucose, sucrose, magnesium carbonate, and the like may beused. The active compound defined above, may be also formulated assuppositories using for example, polyalkylene glycols, for example,propylene glycol, as the carrier. In some embodiments, suppositories areadvantageously prepared from fatty emulsions or suspensions.

The compounds of the present invention can also be administered in theform of liposome delivery systems, such as small unilamellar vesicles,large unilamellar vesicles and multilamellar vesicles. Liposomes can beformed from a variety of phospholipids, containing cholesterol,stearylamine or phosphatidylcholines. In some embodiments, a film oflipid components is hydrated with an aqueous solution of drug to a formlipid layer encapsulating the drug, as described in U.S. Pat. No.5,262,564. For example, the aptamer molecules described herein can beprovided as a complex with a lipophilic compound or non-immunogenic,high molecular weight compound constructed using methods known in theart. Additionally, liposomes may bear aptamers on their surface fortargeting and carrying cytotoxic agents internally to mediate cellkilling. An example of nucleic-acid associated complexes is provided inU.S. Pat. No. 6,011,020.

The compounds of the present invention may also be coupled with solublepolymers as targetable drug carriers. Such polymers can includepolyvinylpyrrolidone, pyran copolymer,polyhydroxypropyl-methacrylamide-phenol,polyhydroxyethylaspanamidephenol, or polyethyleneoxidepolylysinesubstituted with palmitoyl residues. Furthermore, the compounds of thepresent invention may be coupled to a class of biodegradable polymersuseful in achieving controlled release of a drug, for example,polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid,polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates andcross-linked or amphipathic block copolymers of hydrogels.

If desired, the pharmaceutical composition to be administered may alsocontain minor amounts of non-toxic auxiliary substances such as wettingor emulsifying agents, pH buffering agents, and other substances such asfor example, sodium acetate, and triethanolamine oleate.

The dosage regimen utilizing the aptamers is selected in accordance witha variety of factors including type, species, age, weight, sex andmedical condition of the patient; the severity of the condition to betreated; the route of administration; the renal and hepatic function ofthe patient; and the particular aptamer or salt thereof employed. Anordinarily skilled physician or veterinarian can readily determine andprescribe the effective amount of the drug required to prevent, counteror arrest the progress of the condition.

Oral dosages of the present invention, when used for the indicatedeffects, will range between about 0.05 to 7500 mg/day orally. Thecompositions are preferably provided in the form of scored tabletscontaining 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100.0, 250.0,500.0 and 1000.0 mg of active ingredient. Infused dosages, intranasaldosages and transdermal dosages will range between 0.05 to 7500 mg/day.Subcutaneous, intravenous and intraperitoneal dosages will range between0.05 to 3800 mg/day.

Compounds of the present invention may be administered in a single dailydose, or the total daily dosage may be administered in divided doses oftwo, three or four times daily.

Effective plasma levels of the compounds of the present invention rangefrom 0.002 mg/mL to 50 mg/mL. In the dosages of the present invention,mass refers only to the molecular weight of the oligonucleotide portionof the aptamer, irrespective of the mass conferred by PEG conjugation.

Modulation of Pharmacokinetics and Biodistribution of AptamerTherapeutics

It is important that the pharmacokinetic properties for alloligonucleotide-based therapeutics, including aptamers, be tailored tomatch the desired pharmaceutical application. While aptamers directedagainst extracellular targets do not suffer from difficulties associatedwith intracellular delivery (as is the case with antisense andRNAi-based therapeutics), such aptamers must still be able to bedistributed to target organs and tissues, and remain in the body(unmodified) for a period of time consistent with the desired dosingregimen.

Thus, the present invention provides materials and methods to affect thepharmacokinetics of aptamer compositions, and, in particular, theability to tune aptamer pharmacokinetics. The tunability of (i.e., theability to modulate) aptamer pharmacokinetics is achieved throughconjugation of modifying moieties (e.g., PEG polymers) to the aptamerand/or the incorporation of modified nucleotides (e.g., 2′-fluoro or2′-O-methyl) to alter the chemical composition of the nucleic acid. Theability to tune aptamer pharmacokinetics is used in the improvement ofexisting therapeutic applications, or alternatively, in the developmentof new therapeutic applications. For example, in some therapeuticapplications, e.g., in anti-neoplastic or acute care settings whererapid drug clearance or turn-off may be desired, it is desirable todecrease the residence times of aptamers in the circulation.Alternatively, in other therapeutic applications, e.g., maintenancetherapies where systemic circulation of a therapeutic is desired, it maybe desirable to increase the residence times of aptamers in circulation.

In addition, the tunability of aptamer pharmacokinetics is used tomodify the biodistribution of an aptamer therapeutic in a subject. Forexample, in some therapeutic applications, it may be desirable to alterthe biodistribution of an aptamer therapeutic in an effort to target aparticular type of tissue or a specific organ (or set of organs). Inthese applications, the aptamer therapeutic preferentially accumulatesin a specific tissue or organ(s). In other therapeutic applications, itmay be desirable to target tissues displaying a cellular marker or asymptom associated with a given disease, cellular injury or otherabnormal pathology, such that the aptamer therapeutic preferentiallyaccumulates in the affected tissue. For example, as described incopending provisional application U.S. Ser. No. 60/550790, filed on Mar.5, 2004, and entitled “Controlled Modulation of the Pharmacokinetics andBiodistribution of Aptamer Therapeutics), PEGylation of an aptamertherapeutic (e.g., PEGylation with a 20 kDa PEG polymer) is used totarget inflamed tissues, such that the PEGylated aptamer therapeuticpreferentially accumulates in inflamed tissue.

To determine the pharmacokinetic and biodistribution profiles of aptamertherapeutics (e.g., aptamer conjugates or aptamers having alteredchemistries, such as modified nucleotides) a variety of parameters aremonitored. Such parameters include, for example, the half-life(t_(1/2)), the plasma clearance (CL), the volume of distribution (Vss),the area under the concentration-time curve (AUC), maximum observedserum or plasma concentration (C_(max)), and the mean residence time(MRT) of an aptamer composition. As used herein, the term “AUC” refersto the area under the plot of the plasma concentration of an aptamertherapeutic versus the time after aptamer administration. The AUC valueis used to estimate the bioavailability (i.e., the percentage ofadministered aptamer therapeutic in the circulation after aptameradministration) and/or total clearance (CL) (i.e., the rate at which theaptamer therapeutic is removed from circulation) of a given aptamertherapeutic. The volume of distribution relates the plasma concentrationof an aptamer therapeutic to the amount of aptamer present in the body.The larger the Vss, the more an aptamer is found outside of the plasma(i.e., the more extravasation).

The present invention provides materials and methods to modulate, in acontrolled manner, the pharmacokinetics and biodistribution ofstabilized aptamer compositions in vivo by conjugating an aptamer to amodulating moiety such as a small molecule, peptide, or polymer terminalgroup, or by incorporating modified nucleotides into an aptamer. Asdescribed herein, conjugation of a modifying moiety and/or alteringnucleotide(s) chemical composition alter fundamental aspects of aptamerresidence time in circulation and distribution to tissues.

In addition to clearance by nucleases, oligonucleotide therapeutics aresubject to elimination via renal filtration. As such, anuclease-resistant oligonucleotide administered intravenously typicallyexhibits an in vivo half-life of <10 min, unless filtration can beblocked. This can be accomplished by either facilitating rapiddistribution out of the blood stream into tissues or by increasing theapparent molecular weight of the oligonucleotide above the effectivesize cut-off for the glomerulus. Conjugation of small therapeutics to aPEG polymer (PEGylation), described below, can dramatically lengthenresidence times of aptamers in circulation, thereby decreasing dosingfrequency and enhancing effectiveness against vascular targets.

Aptamers can be conjugated to a variety of modifying moieties, such ashigh molecular weight polymers, e.g., PEG; peptides, e.g., Tat (a13-amino acid fragment of the HIV Tat protein (Vives, et al., (1997), J.Biol. Chem. 272(25): 16010-7)), Ant (a 16-amino acid sequence derivedfrom the third helix of the Drosophila antennapedia homeotic protein(Pietersz, et al., (2001), Vaccine 19(11-12): 1397-405)) and Arg₇ (ashort, positively charged cell-permeating peptides composed ofpolyarginine (Arg₇) (Rothbard, et al., (2000), Nat. Med. 6(11): 1253-7;Rothbard, J et al., (2002), J. Med. Chem. 45(17): 3612-8)); and smallmolecules, e.g., lipophilic compounds such as cholesterol. Among thevarious conjugates described herein, in vivo properties of aptamers arealtered most profoundly by complexation with PEG groups. For example,complexation of a mixed 2′ F and 2′-OMe modified aptamer therapeuticwith a 20 kDa PEG polymer hinders renal filtration and promotes aptamerdistribution to both healthy and inflamed tissues. Furthermore, the 20kDa PEG polymer-aptamer conjugate proves nearly as effective as a 40 kDaPEG polymer in preventing renal filtration of aptamers. While one effectof PEGylation is on aptamer clearance, the prolonged systemic exposureafforded by presence of the 20 kDa moiety also facilitates distributionof aptamer to tissues, particularly those of highly perfused organs andthose at the site of inflammation. The aptamer-20 kDa PEG polymerconjugate directs aptamer distribution to the site of inflammation, suchthat the PEGylated aptamer preferentially accumulates in inflamedtissue. In some instances, the 20 kDa PEGylated aptamer conjugate isable to access the interior of cells, such as, for example, kidneycells.

Modified nucleotides can also be used to modulate the plasma clearanceof aptamers. For example, an unconjugated aptamer which incorporatesboth 2′-F and 2′-OMe stabilizing chemistries, which is typical ofcurrent generation aptamers as it exhibits a high degree of nucleasestability in vitro and in vivo, displays rapid loss from plasma (i.e.,rapid plasma clearance) and a rapid distribution into tissues, primarilyinto the kidney, when compared to unmodified aptamer.

PEG-Derivatized Nucleic Acids

As described above, derivatization of nucleic acids with high molecularweight non-immunogenic polymers has the potential to alter thepharmacokinetic and pharmacodynamic properties of nucleic acids makingthem more effective therapeutic agents. Favorable changes in activitycan include increased resistance to degradation by nucleases, decreasedfiltration through the kidneys, decreased exposure to the immune system,and altered distribution of the therapeutic through the body.

The aptamer compositions of the invention may be derivatized withpolyalkylene glycol (PAG) moieties. Examples of PAG-derivatized nucleicacids are found in U.S. patent application Ser. No. 10/718,833, filed onNov. 21, 2003, which is herein incorporated by reference in itsentirety. Typical polymers used in the invention include poly(ethyleneglycol) (PEG), also known as poly(ethylene oxide) (PEO) andpolypropylene glycol (including poly isopropylene glycol). Additionally,random or block copolymers of different alkylene oxides (e.g., ethyleneoxide and propylene oxide) can be used in many applications. In its mostcommon form, a polyalkylene glycol, such as PEG, is a linear polymerterminated at each end with hydroxyl groups:HO—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—OH. This polymer, alpha-,omega-dihydroxylpoly(ethylene glycol), can also be represented asHO-PEG-OH, where it is understood that the—PEG—symbol represents thefollowing structural unit: —CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂— where ntypically ranges from about 4 to about 10,000.

As shown, the PEG molecule is di-functional and is sometimes referred toas “PEG diol.” The terminal portions of the PEG molecule are relativelynon-reactive hydroxyl moieties, the —OH groups, that can be activated,or converted to functional moieties, for attachment of the PEG to othercompounds at reactive sites on the compound. Such activated PEG diolsare referred to herein as bi-activated PEGs. For example, the terminalmoieties of PEG diol have been functionalized as active carbonate esterfor selective reaction with amino moieties by substitution of therelatively nonreactive hydroxyl moieties, —OH, with succinimidyl activeester moieties from N-hydroxy succinimide.

In many applications, it is desirable to cap the PEG molecule on one endwith an essentially non-reactive moiety so that the PEG molecule ismono-functional (or mono-activated). In the case of protein therapeuticswhich generally display multiple reaction sites for activated PEGs,bi-functional activated PEGs lead to extensive cross-linking, yieldingpoorly functional aggregates. To generate mono-activated PEGs, onehydroxyl moiety on the terminus of the PEG diol molecule typically issubstituted with non-reactive methoxy end moiety, —OCH₃. The other,un-capped terminus of the PEG molecule typically is converted to areactive end moiety that can be activated for attachment at a reactivesite on a surface or a molecule such as a protein.

PAGs are polymers which typically have the properties of solubility inwater and in many organic solvents, lack of toxicity, and lack ofimmunogenicity. One use of PAGs is to covalently attach the polymer toinsoluble molecules to make the resulting PAG-molecule “conjugate”soluble. For example, it has been shown that the water-insoluble drugpaclitaxel, when coupled to PEG, becomes water-soluble. Greenwald, etal., J. Org. Chem., 60:331-336 (1995). PAG conjugates are often used notonly to enhance solubility and stability but also to prolong the bloodcirculation half-life of molecules.

Polyalkylated compounds of the invention are typically between 5 and 80kDa in size however any size can be used, the choice dependent on theaptamer and application. Other PAG compounds of the invention arebetween 10 and 80 kDa in size. Still other PAG compounds of theinvention are between 10 and 60 kDa in size. For example, a PAG polymermay be at least 10, 20, 30, 40, 50, 60, or 80 kDa in size. Such polymerscan be linear or branched.

In contrast to biologically-expressed protein therapeutics, nucleic acidtherapeutics are typically chemically synthesized from activated monomernucleotides. PEG-nucleic acid conjugates may be prepared byincorporating the PEG using the same iterative monomer synthesis. Forexample, PEGs activated by conversion to a phosphoramidite form can beincorporated into solid-phase oligonucleotide synthesis. Alternatively,oligonucleotide synthesis can be completed with site-specificincorporation of a reactive PEG attachment site. Most commonly this hasbeen accomplished by addition of a free primary amine at the 5′-terminus(incorporated using a modifier phosphoramidite in the last coupling stepof solid phase synthesis). Using this approach, a reactive PEG (e.g.,one which is activated so that it will react and form a bond with anamine) is combined with the purified oligonucleotide and the couplingreaction is carried out in solution.

The ability of PEG conjugation to alter the biodistribution of atherapeutic is related to a number of factors including the apparentsize (e.g., as measured in terms of hydrodynamic radius) of theconjugate. Larger conjugates (>10 kDa) are known to more effectivelyblock filtration via the kidney and to consequently increase the serumhalf-life of small macromolecules (e.g., peptides, antisenseoligonucleotides). The ability of PEG conjugates to block filtration hasbeen shown to increase with PEG size up to approximately 50 kDa (furtherincreases have minimal beneficial effect as half life becomes defined bymacrophage-mediated metabolism rather than elimination via the kidneys).

Production of high molecular weight PEGs (>10 kDa) can be difficult,inefficient, and expensive. As a route towards the synthesis of highmolecular weight PEG-nucleic acid conjugates, previous work has beenfocused towards the generation of higher molecular weight activatedPEGs. One method for generating such molecules involves the formation ofa branched activated PEG in which two or more PEGs are attached to acentral core carrying the activated group. The terminal portions ofthese higher molecular weight PEG molecules, i.e., the relativelynon-reactive hydroxyl (—OH) moieties, can be activated, or converted tofunctional moieties, for attachment of one or more of the PEGs to othercompounds at reactive sites on the compound. Branched activated PEGswill have more than two termini, and in cases where two or more terminihave been activated, such activated higher molecular weight PEGmolecules are referred to herein as, multi-activated PEGs. In somecases, not all termini in a branch PEG molecule are activated. In caseswhere any two termini of a branch PEG molecule are activated, such PEGmolecules are referred to as bi-activated PEGs. In some cases where onlyone terminus in a branch PEG molecule is activated, such PEG moleculesare referred to as mono-activated. As an example of this approach,activated PEG prepared by the attachment of two monomethoxy PEGs to alysine core which is subsequently activated for reaction has beendescribed (Harris et al., Nature, vol. 2: 214-221, 2003).

The present invention provides another cost effective route to thesynthesis of high molecular weight PEG-nucleic acid (preferably,aptamer) conjugates including multiply PEGylated nucleic acids. Thepresent invention also encompasses PEG-linked multimericoligonucleotides, e.g., dimerized aptamers. The present invention alsorelates to high molecular weight compositions where a PEG stabilizingmoiety is a linker which separates different portions of an aptamer,e.g., the PEG is conjugated within a single aptamer sequence, such thatthe linear arrangement of the high molecular weight aptamer compositionis, e.g., nucleic acid-PEG-nucleic acid (-PEG-nucleic acid)_(n) where nis greater than or equal to 1.

High molecular weight compositions of the invention include those havinga molecular weight of at least 10 kDa. Compositions typically have amolecular weight between 10 and 80 kDa in size. High molecular weightcompositions of the invention are at least 10, 20, 30, 40, 50, 60, or 80kDa in size.

A stabilizing moiety is a molecule, or portion of a molecule, whichimproves pharmacokinetic and pharmacodynamic properties of the highmolecular weight aptamer compositions of the invention. In some cases, astabilizing moiety is a molecule or portion of a molecule which bringstwo or more aptamers, or aptamer domains, into proximity, or providesdecreased overall rotational freedom of the high molecular weightaptamer compositions of the invention. A stabilizing moiety can be apolyalkylene glycol, such a polyethylene glycol, which can be linear orbranched, a homopolymer or a heteropolymer. Other stabilizing moietiesinclude polymers such as peptide nucleic acids (PNA). Oligonucleotidescan also be stabilizing moieties; such oligonucleotides can includemodified nucleotides, and/or modified linkages, such asphosphorothioates. A stabilizing moiety can be an integral part of anaptamer composition, i.e., it is covalently bonded to the aptamer.

Compositions of the invention include high molecular weight aptamercompositions in which two or more nucleic acid moieties are covalentlyconjugated to at least one polyalkylene glycol moiety. The polyalkyleneglycol moieties serve as stabilizing moieties. In compositions where apolyalkylene glycol moiety is covalently bound at either end to anaptamer, such that the polyalkylene glycol joins the nucleic acidmoieties together in one molecule, the polyalkylene glycol is said to bea linking moiety. In such compositions, the primary structure of thecovalent molecule includes the linear arrangement nucleicacid-PAG-nucleic acid. One example is a composition having the primarystructure nucleic acid-PEG-nucleic acid. Another example is a lineararrangement of: nucleic acid-PEG-nucleic acid-PEG-nucleic acid.

To produce the nucleic acid-PEG-nucleic acid conjugate, the nucleic acidis originally synthesized such that it bears a single reactive site(e.g., it is mono-activated). In a preferred embodiment, this reactivesite is an amino group introduced at the 5′-terminus by addition of amodifier phosphoramidite as the last step in solid phase synthesis ofthe oligonucleotide. Following deprotection and purification of themodified oligonucleotide, it is reconstituted at high concentration in asolution that minimizes spontaneous hydrolysis of the activated PEG. Ina preferred embodiment, the concentration of oligonucleotide is 1 mM andthe reconstituted solution contains 200 mM NaHCO₃-buffer, pH 8.3.Synthesis of the conjugate is initiated by slow, step-wise addition ofhighly purified bi-functional PEG. In a preferred embodiment, the PEGdiol is activated at both ends (bi-activated) by derivatization withsuccinimidyl propionate. Following reaction, the PEG-nucleic acidconjugate is purified by gel electrophoresis or liquid chromatography toseparate fully-, partially-, and un-conjugated species. Multiple PAGmolecules concatenated (e.g., as random or block copolymers) or smallerPAG chains can be linked to achieve various lengths (or molecularweights). Non-PAG linkers can be used between PAG chains of varyinglengths.

The 2′-O-methyl, 2′-fluoro and other modified nucleotide modificationsstabilize the aptamer against nucleases and increase its half life invivo. The 3′-3′-dT cap also increases exonuclease resistance. See, e.g.,U.S. Pat. Nos. 5,674,685; 5,668,264; 6,207,816; and 6,229,002, each ofwhich is incorporated by reference herein in its entirety.

PAG-Derivatization of a Reactive Nucleic Acid

High molecular weight PAG-nucleic acid-PAG conjugates can be prepared byreaction of a mono-functional activated PEG with a nucleic acidcontaining more than one reactive site. In one embodiment, the nucleicacid is bi-reactive, or bi-activated, and contains two reactive sites: a5′-amino group and a 3′-amino group introduced into the oligonucleotidethrough conventional phosphoramidite synthesis, for example:3′-5′-di-PEGylation as illustrated in FIG. 2. In alternativeembodiments, reactive sites can be introduced at internal positions,using for example, the 5-position of pyrimidines, the 8-position ofpurines, or the 2′-position of ribose as sites for attachment of primaryamines. In such embodiments, the nucleic acid can have several activatedor reactive sites and is said to be multiply activated. Followingsynthesis and purification, the modified oligonucleotide is combinedwith the mono-activated PEG under conditions that promote selectivereaction with the oligonucleotide reactive sites while minimizingspontaneous hydrolysis. In the preferred embodiment, monomethoxy-PEG isactivated with succinimidyl propionate and the coupled reaction iscarried out at pH 8.3. To drive synthesis of the bi-substituted PEG,stoichiometric excess PEG is provided relative to the oligonucleotide.Following reaction, the PEG-nucleic acid conjugate is purified by gelelectrophoresis or liquid chromatography to separate fully-, partially-,and un-conjugated species.

The linking domains can also have one or more polyalkylene glycolmoieties attached thereto. Such PAGs can be of varying lengths and maybe used in appropriate combinations to achieve the desired molecularweight of the composition.

The effect of a particular linker can be influenced by both its chemicalcomposition and length. A linker that is too long, too short, or formsunfavorable steric and/or ionic interactions with the target willpreclude the formation of complex between aptamer and target. A linker,which is longer than necessary to span the distance between nucleicacids, may reduce binding stability by diminishing the effectiveconcentration of the ligand. Thus, it is often necessary to optimizelinker compositions and lengths in order to maximize the affinity of anaptamer to a target.

All publications and patent documents cited herein are incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any is pertinent prior art, nor does it constituteany admission as to the contents or date of the same. The inventionhaving now been described by way of written description, those of skillin the art will recognize that the invention can be practiced in avariety of embodiments and that the foregoing description and examplesbelow are for purposes of illustration and not limitation of the claimsthat follow.

EXAMPLES Example 1 Aptamer Selection and Sequences Example 1A Selectionof rRfY vWF Domain A1 Aptamers

Selections were performed to identify aptamers that bind to human orrabbit vWF A1 domain using a nucleotide pool consisting of 2′-OH purineand 2′-F pyrimidine nucleotides (rRfY). The selection strategy yieldedhigh affinity aptamers specific for human and rabbit vWF A1 domainswhich had been immobilized on a hydrophobic plate.

Pool Preparation

A DNA template with the sequence5′-GGAGCGCACTCAGCCACNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTTTCGACCTCTCTGCTAGC3′ (SEQ ID NO 8) was synthesized using an ABI EXPEDITE™ DNA synthesizer,and deprotected by standard methods. The series of N's in the DNAtemplate (SEQ ID NO 8) can be any combination of nucleotides and givesrise to the unique sequence region of the resulting aptamers. Thetemplate was amplified with the primers5′-TAATACGACTCACTATAGGAGCGCACTCAGCCAC-3′ (SEQ ID NO 9) and5′-GCTAGCAGAGAGGTCGAAA-3′ (SEQ ID NO 10) and then used as a template forin vitro transcription with T7 RNA polymerase (Y639F). Transcriptionswere, typically, incubated at 37° C. overnight, using 40 mM Tris pH 8.0,40 mM DTT, 1 mM spermidine-HCl, 0.002% TritonX-100, 4% (w/v) PEG-8000,12 mM MgCl₂, 3 mM 2′-F-CTP, 3 mM 2′-F-UTP, 3 mM GTP, 3 mM ATP, 0.5×inorganic pyrophosphatase, and 1× T7 polymerase (Y639F), andapproximately 0.5 μM template DNA.

Selection

For the human vWF A1 domain selection, the first ten rounds wereinitiated by immobilizing 24 pmoles of human vWF A1 domain (SEQ ID NO 4,FIG. 4) to the surface of a Nunc Maxisorp hydrophobic plate (NuncCat.#446612, Rochester, N.Y.) for 1 hour at room temperature in 100 μLof 1× Dulbecco's PBS (Gibco BRL Cat.#14040-133, Carlsbad, Calif.). ForRounds eleven and twelve, 12 pmoles of full length human vWF (SEQ ID NO7, accession number VWHU, available from Calbiochem Cat.#681300, LaJolla, Calif.) were immobilized to the hydrophobic plate. For the rabbitvWF selection, each round was initiated by immobilizing 24 pmoles ofrabbit vWF A1 domain (SEQ ID NO 6: accession numberAAB51555, FIG. 3)under the same conditions as for human vWF A1 domain.

In all cases, after one hour of protein immobilization, the supernatantwas removed and the wells were washed 4 times with 120 μL 1× Dulbecco'sPBS. The protein-immobilized well was then blocked with 100 uL blockingbuffer (1× Dulbecco's PBS with 1% BSA) for 1 hour at room temperature.In Round one, 333 pmoles of pool RNA (2×10¹⁴ unique molecules) wereincubated in 100 μL 1× Dulbecco's PBS in the wells containingBSA-blocked immobilized protein target for 1 hour at room temperature.The supernatant was then removed and the wells were washed 4 times with120 μL 1× Dulbecco's PBS. In later rounds, additional washes were addedto increase the stringency of the positive selection step (see Tables 1and 2). Starting at Round 2 and in all subsequent rounds, two negativeselection steps were included before the positive selection step. First,the pool RNA was incubated for 1 hour at room temperature in anunblocked well to remove any plastic binding sequences from the pool. Inthe second negative selection step, the RNA was transferred to a BSAblocked well (not containing the protein target) for 1 hour at roomtemperature to remove any BSA binding sequences from the pool prior tothe positive selection. Starting at Round 2 and in all subsequentrounds, 0.1 mg/mL tRNA and 0.1 mg/mL salmon sperm DNA were spiked intothe positive selection reaction as non-specific competitors. In allcases, the pool RNA bound to the immobilized protein target was reversetranscribed directly in the selection plate with the addition of RT mix(Round 1:100 uL; Round 2+:50 uL; containing the 3′-primer according toSEQ ID NO 10 and Thermoscript RT (Invitrogen Cat.#11146-016, Carlsbad,Calif.) followed by incubation at 65° C. for 1 hour.

The resulting cDNA was used as a template for PCR (Round 1:500 uL; Round2+:250 uL; containing the 5′-primer according to (SEQ ID NO 9), the3′-primer according to (SEQ ID NO 10), and Taq polymerase (New EnglandBiolabs Cat.# MO267L, Beverly, Mass.)). PCR reactions were done underthe following conditions: a) denaturation step: 94° C. for 2 minutes; b)cycling steps: 94° C. for 30 seconds, 60° C. for 30 seconds, 72° C. for1 minute; c) final extension step: 72° C. for 3 minutes. The cycles wererepeated until sufficient PCR product was generated. The minimum numberof cycles required to generate sufficient PCR product is reported inTables 1 and 2 as the “PCR Threshold”. The amplified pool template DNAwas then isopropanol precipitated and half of the PCR product was usedas template for the transcription of pool RNA for the next round ofselection. The transcribed RNA pool was gel purified using a 10%polyacrylamide gel every third round. When not gel-purified, thetranscribed RNA pool was desalted using two Centri-Spin 10 columns(Princeton Separations Cat.# CS-101, Adelphia, N.J.). In all cases, anequivalent of one-tenth of the total transcription product was carriedforward as the starting pool for the subsequent round of selection.

TABLE 1 Human vWF A1 domain selection conditions using an rRfY poolRound Target Washes PCR Threshold Purification 1 24 pmol hA1 4 × 120 uL16 Desalt (2x) 2 24 pmol hA1 4 × 120 uL 18 Desalt (2x) 3 24 pmol hA1 4 ×120 uL 16 Gel purify 4 24 pmol hA1 8 × 120 uL 15 Desalt (2x) 5 24 pmolhA1 8 × 120 uL 15 Desalt (2x) 6 24 pmol hA1 8 × 120 uL 15 Gel purify 724 pmol hA1 8 × 120 uL 12 Desalt (2x) 8 24 pmol hA1 8 × 120 uL 12 Desalt(2x) 9 24 pmol hA1 8 × 120 uL 10 Gel purify 10 24 pmol hA1 6 × 120 uL;10 Desalt (2x) 2 × 120 uL (15 min. each) 11 12 pmol full 6 × 120 uL; 20Desalt (2x) length vWF 2 × 120 uL (15 min. each) 12 12 pmol full 6 × 120uL; 15 Gel purify length vWF 2 × 120 uL (15 min. each)

TABLE 2 Rabbit vWF A1 domain selection conditions using an rRfY pool PCRRound Target Washes Threshold Purification 1 24 pmol rA1 4 × 120 uL 16Desalt (2x) 2 24 pmol rA1 4 × 120 uL 18 Desalt (2x) 3 24 pmol rA1 4 ×120 uL 16 Gel purify 4 24 pmol rA1 8 × 120 uL 15 Desalt (2x) 5 24 pmolrA1 8 × 120 uL 15 Desalt (2x) 6 24 pmol rA1 8 × 120 uL 15 Gel purify 724 pmol rA1 8 × 120 uL 12 Desalt (2x) 8 24 pmol rA1 8 × 120 uL 12 Desalt(2x) 9 24 pmol rA1 8 × 120 uL 10 Gel purify 10 24 pmol rA1 6 × 120 uL;10 Desalt (2x) 2 × 120 uL (15 min. each) 11 24 pmol rA1 6 × 120 uL; 10Desalt (2x) 2 × 120 uL (15 min. each) 12 24 pmol rA1 6 × 120 uL; 10 Gelpurify 2 × 120 uL (15 min. each)vWF Domain A1 Binding Analysis

The selection progress was monitored using a sandwich filter bindingassay. The 5′-³²P-labeled pool RNA (trace concentration) was incubatedwith either a no target protein control, 100 nM human vWF A1 domain (SEQID NO 4) or 100 nM rabbit vWF A1 domain (SEQ ID No 6) 1× Dulbecco's PBScontaining 0.1 mg/mL tRNA, and 0.1 mg/mL salmon sperm DNA (in a finalvolume of 50 uL) for 30 minutes at room temperature and then applied toa nitrocellulose and nylon filter sandwich in a dot blot apparatus(Schleicher and Schuell, Keene, N.H.). The percentage of pool RNA boundto the nitrocellulose was calculated after Rounds 6, 9, and 12 with athree point screen (100 nM human vWF A1 domain, 100 nM rabbit vWF A1domain, and a no-target control). Pool binding was compared to that ofthe naïve pool RNA (Round 0). The results of the rRfY pool bindinganalyses are found in Table 3.

TABLE 3 vWF A1 domain rRfY selection pool binding assays. Pool 100 nMhuman 100 nM rabbit No Selection Round A1 A1 Protein Naive Pool Round 011.2% 14.3% 10.5% Human vWF A1 Round 6 16.0% 16.9% 13.8% Rabbit vWF A1Round 6 15.2% 17.9% 14.7% Human vWF A1 Round 9 14.7% 14.3% 10.5% RabbitvWF A1 Round 9 13.7% 14.7% 10.1% Human vWF A1 Round 12 31.8% 33.1% 13.2%Rabbit vWF A1 Round 12 24.1% 17.7% 10.3%

When a significant positive ratio of binding of RNA in the presence ofhuman or rabbit vWF A1 domain versus in the absence of protein was seen,the pools were cloned using the TOPO TA cloning kit (Invitrogen,Cat.#45-0641, Carlsbad, Calif.) according to the manufacturer'sinstructions. Round 9 and 12 pool templates were cloned and sequenced(125 total sequences), producing 48 unique clones. All unique cloneswere transcribed, desalted, 5-³²P end-labeled, and assayed in a 3-pointdot blot screen (no protein target control, 100 nM human vWF A1 domain(SEQ ID NO 5, FIG. 3), or 100 nM rabbit vWF A1 domain (SEQ ID NO 6, FIG.3). The data is presented in the third and fourth columns of Table 4below as the ratio of the fraction of the aptamer bound to thenitrocellulose in the presence of the target protein to the fraction ofaptamer bound in the absence of the target protein.

Based on this initial screen, K_(D)s were determined for 12 of the bestvWF dependent binding sequences using the dot blot assay and arereported in column 5 of Table 4 below. For K_(D) determination, aptamerstranscripts were purified on 10% denaturing polyacrylamide gels, 5′ endlabeled with γ-³²P ATP. An 8 point titration of human vWF A1 domain (SEQID NO 5) was used in the dot blot assay (1 uM, 300 nM, 100 nM, 30 nM, 10nM, 3 nM, 1 nM, 0 nM), and K_(D) values were calculated by fitting theequation y=(max/(1+K/protein))+yint using KaleidaGraph (KaleidaGraph v.3.51, Synergy Software) For all dot blot assays used to determine singleclone K_(D)s in the Examples described herein, the target protein, e.g.human vWF A1 domain, is diluted with 1× Dulbecco's PBS buffer whichincludes 0.1 mg/mL BSA and incubated with labeled aptamer for 30 minutesat 24° C. prior to filtration and quantitation.

TABLE 4 Human and rabbit vWF A1 domain rRfY aptamer binding activity*human Screen- Screen- A1 K_(D) Aptamer Human/No Protein Rabbit/NoProtein (nM) (AMX201.B1) (SEQ ND ND 19 ID NO 11) (AMX198.G1) SEQ 1.891.90 45 ID NO 12 (AMX201.H3) SEQ 1.88 1.89 90 ID NO13 (AMX201.B3) SEQ1.69 1.64 ND ID NO 14 (AMX201.G1) SEQ 2.14 2.20 190 ID NO 15 (AMX198.C6)SEQ 3.03 4.62 249 ID NO 16 (AMX201.B11) SEQ 1.55 1.52 ND ID NO 17(AMX201.D10) SEQ 1.59 1.52 ND ID NO 18 (AMX198.C10) SEQ 1.40 3.39 555 IDNO 19 (AMX201.H4) SEQ 1.79 1.86 ND ID NO 20 (AMX201.G9) SEQ 2.06 2.11182 ID NO 21 (AMX201.H11) SEQ 1.75 1.40 ND ID NO 22 (AMX201.C8) SEQ 2.471.50 0.2 ID NO 23 (AMX201.H1) SEQ 2.61 2.46 189 ID NO 24 (AMX198.E11)SEQ 1.03 2.37 1056 ID NO 25 (AMX198.A10) SEQ 1.26 5.74 1860 ID NO 26(AMX201.D4) SEQ 2.23 2.46 ND ID NO 27 (AMX201.D3) SEQ 1.76 1.52 ND ID NO28 (AMX201.A8) SEQ 1.82 1.51 ND ID NO 29 (AMX198.E5) SEQ 1.60 1.56 172ID NO 30 *used human vWF A1 domain SEQ ID NO 5 for aptamer screen andaptamer K_(D)s (ND = not done)

The nucleic acid sequences of the rRfY aptamers characterized in Table 4above are given below. The unique sequence of each aptamer below beginsat nucleotide 18, immediately following the sequence GGAGCGCACTCAGCCAC(SEQ ID NO 221), and runs until it meets the 3′fixed nucleic acidsequence TTTCGACCTCTCTGCTAGC (SEQ ID NO 222).

Unless noted otherwise, individual sequences listed below arerepresented in the 5′ to 3′ orientation and were selected under rRfYSELEX™ conditions wherein the purines (A and G) are 2′-OH (ribo) and thepyrimidines (U and C) are 2′-fluoro.

(AMX201.B1) SEQ ID NO 11GGAGCGCACUCAGCCACAGAGCCCUGAGUGUAUGAUCGCCUAGAUCUAUCGAUGCUUUUUCGACCUCUCUGCUAGC (AMX198.G1) SEQ ID NO 12GGAGCGCACUCAGCCACAACACUAAUGGGGAAAGUUCAAGGAUUCUUGACCGGUGCGUUUCGACCUCUCUGCUAGC (AMX201.H3) SEQ ID NO 13GGAGCGCACUCAGCCACUAACGGUUGAUCUCAGGACUAAAUAGUCAACAAGGAUGCGUUUCGACCUCUCUGCUAGC (AMX201.B3) SEQ ID NO 14GGAGCGCACUCAGCCACAGAGCCCUGAGUGUAUGAUCGCCGAGAUCUAUCGAUGCUUUUUCGACCUCUCUGCUAGC (AMX201.G1) SEQ ID NO 15GGAGCGCACUCAGCCACGCUCGGUGGGGAAAUUUUAGCCUAAUUGGCUACUUGUGCGUUUCGACCUCUCUGCUAGC (AMX198.C6) SEQ ID NO 16GGAGCGCACUCAGCCACGGUGGUCAGUCAGUGAUAUGAUUAAGUUCAGCUGUGGCUGUUUCGACCUCUCUGCUAGC (AMX201.B11) SEQ ID NO 17GGAGCGCACUCAGCCACACCGAGGCUGGAUAUCUACGAGAGGAAGUGCUGCUUGAAUUUCGACCUCUCUGCUAGC (AMX201.D10) SEQ ID NO 18GGAGCGCACUCAGCCACACUGAGGCUGGAUAUCUACGAGAGGAAGUGCUGCUUGGAUUUCGACCUCUCUGCUAGC (AMX198.C10) SEQ ID NO 19GGAGCGCACUCAGCCACUGGUCCUUAGCUAGUUGUACUAGCGACGCGUUCAGGUGGUUUCGACCUCUCUGCUAGC (AMX201.H4) SEQ ID NO 20GGAGCGCACUCAGCCACUAACGGUUGAUCUCAGGACUAAUAGUCAACAAGGAUGCGUUUCGACCUCUCUGCUAGC (AMX201.G9) SEQ ID NO 21GGAGCGCACUCAGCCACUAACGGCUGAUCUCAGGACUAAAUAGUCAACAAGGAUGCGUUUCGACCUCUCUGCUAGC (AMX201.H11) SEQ ID NO 22GGAGCGCACUCAGCCACCCUGUCGUCUUUUGGUAGUCAGCCAAAAGCUAGUUGGUUGUUUCGACCUCUCUGCUAGC (AMX201.C8) (ARC840) SEQ ID NO 23GGAGCGCACU CAGCCACCCUCGCAAG CAUUUUAAGAAUGA CUUGUGC CGCUGGCUG UUUUCGACCUCUCUGCUAGC (AMX201.H1) SEQ ID NO 24GGAGCGCACUCAGCCACUUUACGGUGAAAGUCUCUCGGGGUUCCGAGUUACGGUGCGUUUCGACCUCUCUGCUAGC (AMX198.E11) SEQ ID NO 25GGAGCGCACUCAGCCACGGUAACAUUGUUUCCGGCGAUUCUUUGAACGCCGUCGUGGUUUCGACCUCUCUGCUAGC (AMX198.A10) SEQ ID NO 26GGAGCGCACUCAGCCACCAGUUAUGCUGGCUUUGGUCUUUGACUGUCUGAGUGUUCGUUUCGACCUCUCUGCUAGC (AMX201.D4) SEQ ID NO 27GGAGCGCACUCAGCCACUGGGGCUGAUCUCGCACGAUAGUUCGUGUCAAGGAUGCGUUUUCGACCUCUCUGCUAGC (AMX201.D3) SEQ ID NO 28GGAGCGCACUCAGCCACGCCCACGUCAAAUUAUAGUCUACUUUGAUGUGCCCGUGGUUUCGACCUCUCUGCUAGC (AMX201.A8) SEQ ID NO 29GGAGCGCACUCAGCCACGCUGUACACUGAUGUUGUAACAUGUACCCCCUGGCUGUUUCGACCUCUCUGCUAGC (AMX198.E5) SEQ ID NO 30GGAGCGCACUCAGCCACUUCGACUUUCAUGUCUGAAGUCCCUGCAGUGCGAGAGACGUUUCGACCUCUCUGCUAGC

While not wishing to be bound by any theory, based on the binding datapresented in Table 4 above and the activity in cellular assays presentedin Table 21 below for both the full length aptamers from this SELEX™selection and the minimized aptamer sequences (see Example 2a below) thepredicted generic secondary structure and predicted core nucleic acidsequence required for binding to the vWF target of all embodiments ofthe invention derived from this aptamer selection is depicted in FIG. 10as SEQ ID NO 217 (RNAstructure, Version 4.1, Mathews, D. H.; Disney, M.D.; Childs, J. L.; Schroeder, S. J.; Zuker, M.; and Turner, D. H.,“Incorporating chemical modification constraints into a dynamicprogramming algorithm for prediction of RNA secondary structure,” 2004.Proceedings of the National Academy of Sciences, US, 101, 7287-7292).ARC840 (SEQ ID NO 23) is one example of an aptamer having the sequencedepicted in FIG. 10 wherein the bold, underlined regions shown in thesequence listed above denote required bases.

Example 1B Selection of rRdY vWF Domain A1 Aptamers

Selections were performed to identify aptamers that bind to (1) humanvWF A1 domain, (2) rabbit vWF A1 domain, or (3) human and rabbit vWF A1domains using a nucleotide pool consisting of 2′-OH purine anddeoxy-pyrimidine nucleotides (rRdY). The selection strategy yielded highaffinity aptamers specific for human and rabbit vWF A1 domains which hadbeen immobilized on a hydrophobic plate.

Pool Preparation

A DNA template with the sequence5′-GGAGCGCACTCAGCCACNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTTTCGACCTCTCTGCTAGC3′ (SEQ ID NO 8) was synthesized using an ABI EXPEDITE™ DNA synthesizer,and deprotected by standard methods. The series of N's in the DNAtemplate (SEQ ID NO 8) can be any combination of nucleotides and givesrise to the unique sequence region of the resulting aptamers. Thetemplate was amplified with the primers5′-TAATACGACTCACTATAGGAGCGCACTCAGCCAC-3′ (SEQ ID NO 9) and5′-GCTAGCAGAGAGGTCGAAA-3′ (SEQ ID NO 10) and then used as a template forin vitro transcription with T7 RNA polymerase (Y639F). Transcriptionswere, typically, incubated at 37° C. overnight, using 40 mM Tris pH 8.0,40 mM DTT, 1 mM spermidine-HCl, 0.002% Triton X-100, 4% (w/v) PEG-8000,12 mM MgCl₂, 3 mM dCTP, 3 mM dTTP, 3 mM rGTP, 3 mM rATP, 0.5× inorganicpyrophosphatase, and 1× T7 polymerase (Y639F), and approximately 0.5 μMtemplate DNA.

Selection

For the human vWF selection, the first ten rounds were initiated byimmobilizing 24 pmoles of human vWF A1 domain (SEQ ID NO 4) to thesurface of a Nunc Maxisorp hydrophobic plate (Nunc, Cat.#446612Rochester, N.Y.) for 1 hour at room temperature in 100 μL of 1×Dulbecco's PBS (Gibco BRL Cat.#14040-133, Carlsbad, Calif.). For Roundseleven and twelve, 12 pmoles of full length human vWF (SEQ ID NO 7, FIG.4) were immobilized to the hydrophobic plate. For the rabbit vWFselection, each round was initiated by immobilizing 24 pmoles of rabbitvWF A1 domain (SEQ ID NO 6) under the same conditions. For the first tworounds of the human/rabbit alternating selection 12 pmoles of human vWFA1 domain (SEQ ID NO 4) and 12 pmoles of rabbit vWF A1 domain (SEQ ID NO6) were immobilized to a hydrophobic plate as previously described. Inthe subsequent rounds of the alternating selection, the protein targetwas alternated each round between the human and rabbit vWF A1 domain(SEQ ID NOS 4 and 5, respectively), except in round 11, human fulllength vWF (SEQ ID NO 7) was used.

In all cases, after one hour of protein immobilization, the supernatantwas removed and the wells were washed 4 times with 120 μL 1× Dulbecco'sPBS. The protein-immobilized well was then blocked with 100 uL blockingbuffer (1× Dulbecco's PBS with 1% BSA) for 1 hour at room temperature.In Round one, 333 pmoles of pool RNA (2×10¹⁴ unique molecules) wereincubated in 100 μL 1× Dulbecco's PBS in the wells containingBSA-blocked immobilized protein target for 1 hour at room temperature.The supernatant was then removed and the wells were washed 4 times with120 μL 1× Dulbecco's PBS. In later rounds, additional washes were addedto increase the stringency of the positive selection step (see Tables 5,6, and 7). Starting at Round 2 and in all subsequent rounds, twonegative selection steps were included before the positive selectionstep. First, the pool RNA was incubated for 1 hour at room temperaturein an unblocked well to remove any plastic binding sequences from thepool. In the second negative selection step, the RNA was transferred toa BSA blocked well (not containing the protein target) for 1 hour atroom temperature to remove any BSA binding sequences from the pool priorto the positive selection. Starting at Round 2 and in all subsequentrounds, 0.1 mg/mL tRNA and 0.1 mg/mL salmon sperm DNA were spiked intothe positive selection reaction as non-specific competitors.

In all cases, the pool RNA bound to the immobilized protein target wasreverse transcribed directly in the selection plate with the addition ofRT mix (Round 1:100 uL; Round 2+:50 uL; containing the 3′-primeraccording to (SEQ ID NO 10) and Thermoscript RT (Invitrogen,Cat.#11146-016, Carlsbad, Calif.) followed by incubation at 65° C. for 1hour. The resulting cDNA was used as a template for PCR (Round 1:500 uL;Round 2+:250 uL: containing the 5′-primer according to (SEQ ID NO 9),the 3′-primer according to (SEQ ID NO 10), and Taq polymerase (NewEngland Biolabs, Cat.# MO267L, Beverly, Mass.)). PCR reactions were doneunder the following conditions: a) denaturation step: 94° C. for 2minutes; b) cycling steps: 94° C. for 30 seconds, 60° C. for 30 seconds,72° C. for 1 minute; c) final extension step: 72° C. for 3 minutes. Thecycles were repeated until sufficient PCR product was generated. Theminimum number of cycles required to generate sufficient PCR product isreported in Tables 5, 6 and 7 as the “PCR Threshold”. The amplified pooltemplate DNA was then isopropanol precipitated and half of the PCRproduct was used as template for the transcription of pool RNA for thenext round of selection. The transcribed RNA pool was gel purified usinga 10% polyacrylamide gel every two rounds. When not gel-purified, thetranscribed pool was desalted using two Centri-Spin 10 columns(Princeton Separations Cat.# CS-101, Adelphia, N.J.). In all cases, anequivalent of one-tenth of the total transcription product was carriedforward as the starting pool for the subsequent round of selection.

TABLE 5 Human vWF A1 domain selection conditions using an rRdY pool PCRRound Target Washes Threshold Purification 1 24 pmol hA1 4 × 120 uL 13Desalt (2x) 2 24 pmol hA1 4 × 120 uL 18 Desalt (2x) 3 24 pmol hA1 4 ×120 uL 16 Gel purify 4 24 pmol hA1 8 × 120 uL 15 Desalt (2x) 5 24 pmolhA1 8 × 120 uL 15 Desalt (2x) 6 24 pmol hA1 8 × 120 uL 15 Gel purify 724 pmol hA1 8 × 120 uL 12 Desalt (2x) 8 24 pmol hA1 8 × 120 uL 12 Desalt(2x) 9 24 pmol hA1 8 × 120 uL 10 Gel purify 10 24 pmol hA1 6 × 120 uL 10Desalt (2x) 2 × 120 uL (15 min. each) 11 12 pmol full length 6 × 120 uL;20 Desalt (2x) vWF 2 × 120 uL (15 min. each) 12 12 pmol full length 6 ×120 uL; 20 Gel purify vWF 2 × 120 uL (15 min. each)

TABLE 6 Rabbit vWF A1 domain selection conditions using an rRdY poolRound Target Washes PCR Threshold Purification 1 24 pmol rA1 4 × 120 uL13 Desalt (2x) 2 24 pmol rA1 4 × 120 uL 18 Desalt (2x) 3 24 pmol rA1 4 ×120 uL 10 Gel purify 4 24 pmol rA1 8 × 120 uL 15 Desalt (2x) 5 24 pmolrA1 8 × 120 uL 15 Desalt (2x) 6 24 pmol rA1 8 × 120 uL 15 Gel purify 724 pmol rA1 8 × 120 uL 15 Desalt (2x) 8 24 pmol rA1 8 × 120 uL 12 Desalt(2x) 9 24 pmol rA1 8 × 120 uL 10 Gel purify 10 24 pmol rA1 6 × 120 uL;10 Desalt (2x) 2 × 120 uL (15 min. each) 11 24 pmol rA1 6 × 120 uL; 10Desalt (2x) 2 × 120 uL (15 min. each) 12 24 pmol rA1 6 × 120 uL; 10 Gelpurify 2 × 120 uL (15 min. each)

TABLE 7 Human/rabbit vWF A1 domain alternating selection conditionsusing an rRdY pool PCR Round Target Washes Threshold Purification 1 12pmol hA1/ 4 × 120 uL 13 Desalt (2x) 12 pmol rA1 2 12 pmol hA1/ 4 × 120uL 18 Desalt (2x) 12 pmol rA1 3 24 pmol hA1 4 × 120 uL 16 Gel purify 424 pmol rA1 8 × 120 uL 15 Desalt (2x) 5 24 pmol hA1 8 × 120 uL 15 Desalt(2x) 6 24 pmol rA1 8 × 120 uL 15 Gel purify 7 24 pmol hA1 8 × 120 uL 12Desalt (2x) 8 24 pmol rA1 8 × 120 uL 12 Desalt (2x) 9 24 pmol hA1 8 ×120 uL 10 Gel purify 10 24 pmol rA1 6 × 120 uL; 10 Desalt (2x) 2 × 120uL (15 min. each) 11 12 pmol full length 6 × 120 uL; 20 Desalt (2x) vWF2 × 120 uL (15 min. each) 12 24 pmol rA1 6 × 120 uL; 10 Gel purify 2 ×120 uL (15 min. each)vWF Binding Analysis

The selection progress was monitored using a sandwich filter bindingassay. The 5′-³²P-labeled pool RNA (trace concentration) was incubatedwith either a no target protein control, 100 nM human vWF A1 domain or100 nM rabbit vWF A1 domain, in 1× Dulbecco's PBS containing 0.1 mg/mLtRNA, and 0.1 mg/mL salmon sperm DNA (in a final volume of 50 uL) for 30minutes at room temperature and then applied to a nitrocellulose andnylon filter sandwich in a dot blot apparatus (Schleicher and Schuell,Keene, N.H.). The percentage of pool RNA bound to the nitrocellulose wascalculated after Rounds 6, 9, and 12 with a three point screen (100 nMhuman vWF A1 domain, 100 nM rabbit vWF A1 domain, and a no-targetcontrol). Pool binding was compared to that of the naïve pool RNA (Round0). The results of the rRdY pool binding analyses are found in Table 8.

TABLE 8 vWF A1 domain rRdY selection pool binding assays. Selection PoolRound 100 nM hA1 100 nM rA1 No Protein Naïve Pool Round 0 9.7% 10.4%10.5% Human vWF A1 Round 6 19.6% 19.7% 15.3% Rabbit vWF A1 Round 6 14.3%14.4% 12.3% hA1/rA1 Round 6 19.8% 19.8% 15.9% Human vWF A1 Round 9 23.8%24.3% 15.6% Rabbit vWF A1 Round 9 24.4% 24.0% 16.6% hA1/rA1 Round 919.6% 19.4% 14.6% Human vWF A1 Round 12 25.8% 23.0% 17.0% Rabbit vWF A1Round 12 20.7% 20.5% 13.8% hA1/rA1 Round 12 25.2% 26.3% 16.8%

When a significant positive ratio of binding of RNA in the presence ofhuman or rabbit vWF A1 domain (SEQ ID NOS 4 and 6, respectively) versusin the absence of protein was seen, the pools were cloned using the TOPOTA cloning kit (Invitrogen Cat.#45-0641, Carlsbad, Calif.) according tothe manufacturer's instructions. Round 9 and 12 pool templates werecloned and sequenced (185 total sequences), producing 78 unique cloneswithin 3 sequence families. All unique clones were transcribed,desalted, 5-³²P end-labeled, and assayed in a 3-point dot blot screen(no protein target control, 100 nM human vWF A1 domain (SEQ ID NO 5), or100 nM rabbit vWF A1 domain (SEQ ID NO 6). The data are presented in thethird and fourth columns of Table 9 below as the ratio of the fractionof the aptamer bound to the nitrocellulose in the presence of the targetprotein to the fraction of aptamer bound in the absence of the targetprotein. Of the three sequence families, members of Family #1 and #2 andtwo individual, non-family aptamers, bound to both human vWF domain A1(SEQ ID NO 5) and rabbit vWF domain A1 (SEQ ID NO 6).

Based on this initial screen, K_(D)'s were determined for 16 of the bestvWF dependent binding sequences using the dot blot assay. For K_(D)determination, aptamers were purified on denaturing polyacrylamide gelsand 5′-end labeled with γ-³²P ATP. A 6 point protein titration of humanvWF A1 domain (SEQ ID NO 5) was used in the dot blot assay (333 nM, 100nM, 33 nM, 10 nM, 3 nM, 0 nM) in 1× DPBS plus 0.1 mg/mL BSA at roomtemperature for 30 minutes. K_(D) values were calculated by fitting theequation y=(max/(1+K/protein))+yint using KaleidaGraph (KaleidaGraph v.3.51, Synergy Software).

Results of protein binding characterization are tabulated in the finalcolumn of Table 9 below.

TABLE 9 Human and rabbit vWF A1 domain rRdY aptamer binding activity*Screen- Screen- Human/No Rabbit/No Human A1 # Aptamer Protein ProteinK_(D) (nM) 1 (AMX203.D6) SEQ ID 1.92 1.57 523 NO 31 2 (AMX205.H8) SEQ ID2.04 2.84 788 NO 32 3 (AMX205.H11) SEQ ID 2.18 2.41 144 NO 33 4(AMX205.A7) SEQ ID 1.24 1.37 ND NO 34 5 (AMX205.D11) SEQ ID 2.22 2.07124 NO 35 6 (AMX206.F9) SEQ ID 2.98 3.00 139 NO 36 7 (AMX206.H9) SEQ ID1.98 2.31 109 NO 37 8 (AMX206.A10) SEQ ID 2.62 2.58 111 NO 38 9(AMX205.F9) SEQ ID 2.22 2.47 145 NO 39 10 (AMX206.E7) SEQ ID 2.11 2.26151 NO 40 11 (AMX206.D7) SEQ ID 2.19 2.08 187 NO 41 12 (AMX203.A6) SEQID 1.16 1.16 ND NO 42 13 (AMX203.A1) SEQ ID 2.99 2.67 1148  NO 43 14(AMX203.G9) SEQ ID 1.65 1.35    1.3 NO 44 15 (AMX205.H9) SEQ ID 2.363.14 178 NO 45 16 (AMX206.D8) SEQ ID 2.80 3.76 370 NO 46 17 (AMX203.F9)SEQ ID 1.45 1.29 ND NO 47 18 (AMX205.G9) SEQ ID 1.30 1.73 ND NO 48 19(AMX205.F7) SEQ ID 3.13 2.37    1.5 NO 49 20 (AMX205.H10) SEQ ID 1.882.47 397 NO 50 *used human vWF A1 domain (SEQ ID NO 5) for aptamerscreen and aptamer K_(D)s ND = not done

The nucleic acid sequences of the rRdY aptamers characterized in Table 9above are described below. The unique sequence of each aptamer belowbegins at nucleotide 18, immediately following the sequenceGGAGCGCACTCAGCCAC (SEQ ID NO 221), and runs until it meets the 3′fixednucleic acid sequence TTTCGACCTCTCTGCTAGC (SEQ ID NO 222).

Unless noted otherwise, individual sequences listed below arerepresented in the 5′ to 3′ orientation and were selected under rRdYSELEX™ conditions wherein adenosine triphosphate and guanosinetriphosphate are 2′-OH and cytidine triphosphate and thymidinetriphosphate are deoxy.

vWF rRdY SELEX™ Family #1

The core target protein binding motifs for vWF rRdY Family #1 are shownin bold and underlined in the sequences below:

(AMX203.G9) (ARC842) SEQ ID NO 44GGAGCGCACT CAGCCACGGGGTGGGTAGACGGCGGGTATGTGGCTG GTGTCGAAGGGTTTCGACCTCTCTGCTAGC (AMX203.F9) SEQ ID NO 47GGAGCGCACTCAGC CACTGAAGGGTAAGGACGAGGAGGGTATACAGTG TGCGCGTGTATTTCGACCTCTCTGCTAGC (AMX203.A6) SEQ ID NO 42GGAGCGCACTCA GCCACCACGGGGACGGGTAGGGCGGGCGAGGTGGTGG   C ATTAGCGTTTCGACCTCTCTGCTAGC

The predicted secondary structure and core nucleic acid sequencesrequired for binding to the vWF target of some embodiments of theinvention is depicted in FIG. 12 as SEQ ID NO 218.

(AMX203.D6) SEQ ID NO 31GGAGCGCACTCAGCCACAGTTCTGTCGGTGATGAATTAGCGCGAGAGCTGTGGGACGTTTCGACCTCTCTGCTAGC (AMX205.H8), SEQ ID NO 32GGAGCGCACTCAGCCACAAACGGACGGTGATGGATTAACGCGGGTTTATGGCAAGGTTTCGACCTCTCTGCTAGC (AMX205.H11), SEQ ID NO 33GGAGCGCACTCAGCCACGGCACGACGGTGATGGATTAGCGCGGTGTCGGTGGTGTCATTTCGACCTCTCTGCTAGC (AMX205.D11), SEQ ID NO 35GGAGCGCACTCAGCCACGGCACGACGGTGATGAATTAGCGCGGTGTCGGTGGTGTCATTTCGACCTCTCTGCTAGC (AMX206.F9), SEQ ID NO 36GGAGCGCACTCAGCCACGGAGCGTCGGTGATGGATTAGCGCGGCTCCGTGGTACACATTTCGACCTCTCTGCTAGC (AMX206.H9), SEQ ID NO 37GGAGCGCACTCAGCCACGGAGCGTCGGTGATGGATTAGCGCGGTTCCGTGGTACACCTTTCGACCTCTCTGCTAGC (AMX206.A10), SEQ ID NO 38GGAGCGCACTCAGCCACGGCATGACGGTGATGAATTAGCGCGGTGTCGGTGGTGTCATTTCGACCTCTCTGCTAGC (AMX205.F9), SEQ ID NO 39GGAGCGCACTCAGCCACGGAGCGTCGGTGATGGATTAGCGCGGCTCCGTGGTACGCCTTTCGACCTCTCTGCTAGC (AMX206.E7), SEQ ID NO 40GGAGCGCACTCAGCCACGGAGCGTCGGTGATGGATTAGCGCGGCTCCGTGGTACACCTTTCGACCTCTCTGCTAGC (AMX206.D7), SEQ ID NO 41GGAGCGCACTCAGCCACGGCACGACGGTGATGAATTAGCGCGGTGTCGGTGGTGTTATTTCGACCTCTCTGCTAGC (AMX203.A1), SEQ ID NO 43GGAGCGCACTCAGCCACAGTTCTGTCGGTGATGAATTAGCGCGGGAGCTGTGGGACGTTTCGACCTCTCTGCTAGC (AMX205.H9), SEQ ID NO 45GGAGCGCACTCAGCCACGACGGTGATGGATTAGCGCGGTGGAGAAGATGCGCTGTTGTTTCGACCTCTCTGCTAGC (AMX206.D8), SEQ ID NO 46GGAGCGCACTCAGCCACGACGGTGATGGATTAGCGCGGTGGATCTTAACGTGCGAGTTTCGACCTCTCTGCTAGC (AMX205.G9), SEQ ID NO 48GGAGCGCACTCAGCCACAACTGGTTGTCGGTGATGGCATTAACGCGGACCAGGCATGTTTCGACCTCTCTGCTAGC (AMX205.H10), SEQ ID NO 50GGAGCGCACTCAGCCACTGTTGCCGACGGTGATGTATTAACGCGGGCAACGTTGGTGTTTCGACCTCTCTGCTAGCvWF rRdY SELEX™ Single Sequences

The predicted core nucleic acid binding motif for SEQ ID NO 49 is shownin bold and underlined below:

(AMX205.F7) (ARC 841) SEQ ID NO 49GGAGCGCACTCAGCCACACGACATTGGCGGGTTGTAATTACCACGCATGGCTGTTTGTTTCGACCTCTCTGCTAGC (AMX205.A7), SEQ ID NO 34GGAGCGCACTCAGCCACTCAAGGGGGTCGCGTGGGGACGAAGGGTTGCAGTGTGTCGTTTCGACCTCTCTGCTAGC

The predicted core nucleic acid sequences and secondary structurerequired for binding to the vWF target of some embodiments of theinvention is depicted in FIG. 13 as SEQ ID NO 219.

Example 1C Selection #1 of DNA vWF Domain A1 Aptamers

Selections were performed to identify aptamers that bind to (1) humanvWF A1 domain, (2) rabbit vWF A1 domain, or (3) human and rabbit vWF A1domains, using a nucleotide pool consisting of deoxy-nucleotides (DNA).The selection strategy yielded high affinity aptamers specific for humanand rabbit vWF A1 domains which had been immobilized on a hydrophobicplate.

Pool Preparation

A DNA template with the sequence5′-CTACCTACGATCTGACTAGCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGCTTACTCTCATGTAGTTCC-3′(SEQ ID NO 51) (ARC 493) was synthesized using an ABI EXPEDITE™ DNAsynthesizer, and deprotected by standard methods. The series of N's inthe DNA template (SEQ ID NO 51) can be any combination of nucleotidesand gives rise to the unique sequence region of the resulting aptamers.The template was PCR amplified with the primers(5′-CTACCTACGATCTGACTAGC-3′) (SEQ ID NO 52) and(5′-AGGAACTACATGAGAGTAAGC(OH)-3′) (SEQ ID NO 53) under standardconditions. The PCR product was subjected to alkaline hydrolysis (333 mMNaOH, 90° C., 15 min) followed by precipitation. The strands wereseparated on a 10% denaturing polyacrylamide gel and the single strandedDNA pool, which migrated with a lower mobility, was excised from thegel, passively eluted, and precipitated with isopropanol.

Selection

For the human vWF selection, the first ten rounds were initiated byimmobilizing 24 pmoles of human vWF A1 domain (SEQ ID NO 4) to thesurface of a Nunc Maxisorp hydrophobic plate (Nunc, Cat.#446612,Rochester, N.Y.) for 1 hour at room temperature in 100 μL of 1×Dulbecco's PBS (Gibco BRL, Cat.#14040-133, Carlsbad, Calif.). For Roundseleven and twelve, 12 pmoles of full length human vWF (SEQ ID NO 7) wereimmobilized to the hydrophobic plate. For the rabbit vWF selection, eachround was initiated by immobilizing 24 pmoles of rabbit vWF A1 (SEQ IDNO 6) domain under the same conditions. For the first two rounds of thehuman/rabbit alternating selection, 12 pmoles of human vWF A1 domain(SEQ ID NO 4) and 12 pmoles of rabbit vWF A1 domain (SEQ ID NO 6) wereimmobilized to a hydrophobic plate as previously described. In thesubsequent rounds of the alternating selection, the protein target wasalternated each round between human and rabbit vWF A1 domain, except inRound 11 human full length vWF (SEQ ID NO 7) was used.

In all cases, after one hour of protein immobilization, the supernatantwas removed and the wells were washed 4 times with 120 μL 1× Dulbecco'sPBS. The protein-immobilized well was then blocked with 100 uL blockingbuffer (1× Dulbecco's PBS with 1% BSA) for 1 hour at room temperature.In Round one, 333 pmoles of pool DNA (2×10¹⁴ unique molecules) wereincubated in 100 μL 1× Dulbecco's PBS in the wells containingBSA-blocked immobilized protein target for 1 hour at room temperature.The supernatant was then removed and the wells were washed 4 times with120 μL 1× Dulbecco's PBS. In later rounds, additional washes were addedto increase the stringency of the positive selection step (see Tables10, 11, and 12). Starting at Round 2 and in all subsequent rounds, twonegative selection steps were included before the positive selectionstep. First, the pool DNA was incubated for 1 hour at room temperaturein an unblocked well to remove any plastic binding sequences from thepool. In the second negative selection step, the DNA was transferred toa BSA blocked well (not containing the protein target) for 1 hour atroom temperature to remove any BSA binding sequences from the pool priorto the positive selection. Starting at Round 2 and in all subsequentrounds, 0.1 mg/mL tRNA and 0.1 mg/mL salmon sperm DNA were spiked intothe positive selection reaction as non-specific competitors.

In all cases, the pool DNA bound to the immobilized protein target waseluted with 2×100 μL washes with elution buffer (preheated to 90° C., 7M Urea, 100 mM NaOAc pH 5.3, 3 mM EDTA) for five minutes. Both elutionswere pooled and precipitated by the addition of ethanol, then amplifiedin an initial PCR reaction (100 μL reactions including the 5′-primeraccording to SEQ ID NO 52, and the 3′-primer according to SEQ ID NO 53,and Taq polymerase (New England BioLabs, Cat.# M0267L, Beverly, Mass.).PCR reactions were done under the following conditions: a) denaturationstep: 94° C. for 2 minutes; b) cycling steps: 94° C. for 30 seconds, 52°C. for 30 seconds, 72° C. for 1 minute; c) final extension step: 72° C.for 3 minutes. The cycles were repeated until sufficient PCR product wasgenerated. The minimum number of cycles required to generate sufficientPCR product is reported in Tables 10, 11 and 12 as the “PCR Threshold”.10 μL of the PCR product was added to another 300 μL of PCR mix for aprep-scale PCR reaction. The prep-scale PCR product was ethanolprecipitated and was subjected to alkaline hydrolysis (333 mM NaOH, 90°C., 15 min). The strands were separated on a 10% denaturingpolyacrylamide gel and the single stranded DNA pool, which migrated witha lower mobility, was excised from the gel, passively eluted, andprecipitated with isopropanol. In all cases, an equivalent of half ofthe total single stranded DNA product was carried forward as thestarting pool for the subsequent round of selection.

TABLE 10 Human vWF A1 domain selection conditions using a DNA pool PCRRound Target Washes Threshold Purification 1 24 pmol hA1 4 × 120 uL 10Gel purify 2 24 pmol hA1 4 × 120 uL 15 Gel purify 3 24 pmol hA1 4 × 120uL 13 Gel purify 4 24 pmol hA1 8 × 120 uL 15 Gel purify 5 24 pmol hA1 8× 120 uL 15 Gel purify 6 24 pmol hA1 8 × 120 uL 20 Gel purify 7 24 pmolhA1 8 × 120 uL 10 Gel purify 8 24 pmol hA1 8 × 120 uL 10 Gel purify 9 24pmol hA1 8 × 120 uL 10 Gel purify 10 24 pmol hA1 6 × 120 uL; 12 Gelpurify 2 × 120 uL (15 min. each) 11 12 pmol full length 6 × 120 uL; 18Gel purify vWF 2 × 120 uL (15 min. each) 12 12 pmol full length 6 × 120uL; 15 Gel purify vWF 2 × 120 uL (15 min. each)

TABLE 11 Rabbit vWF A1 domain selection conditions using a DNA pool PCRRound Target Washes Threshold Purification 1 24 pmol rA1 4 × 120 uL 10Gel purify 2 24 pmol rA1 4 × 120 uL 13 Gel purify 3 24 pmol rA1 4 × 120uL 13 Gel purify 4 24 pmol rA1 8 × 120 uL 15 Gel purify 5 24 pmol rA1 8× 120 uL 15 Gel purify 6 24 pmol rA1 8 × 120 uL 20 Gel purify 7 24 pmolrA1 8 × 120 uL 10 Gel purify 8 24 pmol rA1 8 × 120 uL 10 Gel purify 9 24pmol rA1 8 × 120 uL 10 Gel purify 10 24 pmol rA1 6 × 120 uL; 12 Gelpurify 2 × 120 uL (15 min. each) 11 24 pmol rA1 6 × 120 uL; 10 Gelpurify 2 × 120 uL (15 min. each) 12 24 pmol rA1 6 × 120 uL; 10 Gelpurify 2 × 120 uL (15 min. each)

TABLE 12 Human/rabbit vWF A1 domain alternating selection conditionsusing a DNA pool PCR Round Target Washes Threshold Purification 1 12pmol hA1/ 4 × 120 uL 12 Gel purify 12 pmol rA1 2 12 pmol hA1/ 4 × 120 uL15 Gel purify 12 pmol rA1 3 24 pmol hA1 4 × 120 uL 10 Gel purify 4 24pmol rA1 8 × 120 uL 15 Gel purify 5 24 pmol hA1 8 × 120 uL 15 Gel purify6 24 pmol rA1 8 × 120 uL 20 Gel purify 7 24 pmol hA1 8 × 120 uL 12 Gelpurify 8 24 pmol rA1 8 × 120 uL 12 Gel purify 9 24 pmol hA1 8 × 120 uL12 Gel purify 10 24 pmol rA1 6 × 120 uL; 12 Gel purify 2 × 120 uL (15min. each) 11 12 pmol full length 6 × 120 uL; 18 Gel purify vWF 2 × 120uL (15 min. each) 12 24 pmol rA1 6 × 120 uL; 10 Gel purify 2 × 120 uL(15 min. each)vWF Binding Analysis

The selection progress was monitored using a sandwich filter bindingassay. The 5′-³²P-labeled pool DNA (trace concentration) was incubatedwith either a no target protein control, 100 nM human vWF A1 domain (SEQID NO 4), or 100 nM rabbit vWF A1 domain (SEQ ID No 6), in 1× Dulbecco'sPBS containing 0.1 mg/mL tRNA, and 0.1 mg/mL salmon sperm DNA in a(final volume of 50 uL) for 30 minutes at room temperature and thenapplied to a nitrocellulose and nylon filter sandwich in a dot blotapparatus (Schleicher and Schuell, Keene, N.H.) The percentage of poolDNA bound to the nitrocellulose was calculated after Rounds 6, 9, and 12with a three point screen (no protein target control, 100 nM human vWFA1 domain (SEQ ID NO 5), 100 nM rabbit vWF A1 domain (SEQ ID NO 6). Poolbinding was compared to that of the naïve pool DNA (Round 0). Theresults of the DNA pool binding analyses are found in Table 13.

TABLE 13 vWF A1 domain DNA selection pool binding assays. Selection PoolRound 100 nM hA1 100 nM rA1 No Protein Naïve Pool Round 0 30.1% 35.4%29.4% Human vWF A1 Round 6 34.4% 36.4% NA Rabbit vWF A1 Round 6 37.9%36.8% 35.6% hA1/rA1 Round 6 47.9% 50.6% 49.0% Human vWF A1 Round 9 30.4%43.7% 19.1% Rabbit vWF A1 Round 9 15.9% 35.0%  6.6% hA1/rA1 Round 940.8% 49.5% 34.7% Human vWF A1 Round 12 36.7% 45.9% 33.0% Rabbit vWF A1Round 12 23.7% 38.7% 13.4% hA1/rA1 Round 12 21.4% 34.2% 16.4%

When a significant positive ratio of binding of DNA in the presence ofhuman or rabbit vWF A1 domain versus in the absence of protein was seen,the pools were cloned using the TOPO TA cloning kit (Invitrogen,Carlsbad, Calif., Cat.#45-0641) according to the manufacturer'sinstructions. Round 9 and 12 pool templates were cloned and sequenced(243 total sequences), producing 106 unique clones within 8 sequencefamilies, 41 of which bound to the vWF target and fell into the familiesdescribed below.

All unique clones were assayed in a 3-point dot blot screen (no proteintarget control, 100 nM human vWF A1 domain (SEQ ID NO 5), or 100 nMrabbit vWF A1 domain (SEQ ID NO 6). The data are presented in the thirdand fourth columns of Table 14 below as the ratio of the fraction of theaptamer bound to the nitrocellulose in the presence of the targetprotein to the fraction of aptamer bound in the absence of the targetprotein.

Based on this initial screen, K_(D)'s were determined for 10 of the vWFdependent binding sequences. For K_(D) determination, aptamers were5′end labeled with γ-³²P ATP and a competition dot blot assay was usedwith a constant protein concentration of 100 nM and an 8 point coldcompetitor DNA titration (333 nM, 100 nM, 33 nM, 10 nM, 3 nM, 1 nM, 33pM, 0 pM) in 1× DPBS plus 0.1 mg/mL BSA at room temperature for 30minutes. K_(D) values were calculated by fitting the equationy=(max/(1+K/protein))+yint using KaleidaGraph (KaleidaGraph v. 3.51,Synergy Software). Results of protein binding characterization aretabulated in Table 14.

TABLE 14 Human and rabbit vWF A1 domain DNA aptamer binding activity*Human Screen-Human/ Screen- A1 K_(D) # Aptamer No Protein Rabbit/NoProtein (nM) 1 1 (AMX199.B3) SEQ 1.76 1.99 30 ID NO 54 2 (AMX199.D11)4.19 3.42 30 SEQ ID NO 55 3 (AMX200.G11) 1.73 1.28 ND SEQ ID NO 56 4(AMX200.D11) 3.34 2.04 ND SEQ ID NO 57 5 (AMX200.D8) SEQ 2.86 1.61 ND IDNO 58 6 (AMX200.C11) SEQ 6.00 3.64 26 ID NO 59 7 (AMX199.G10) 4.97 2.6622 SEQ ID NO 60 8 (AMX199.F7) SEQ 5.47 5.00 22 ID NO 61 9 (AMX200.A7)SEQ 0.97 0.96 ND ID NO 62 10 (AMX200.B9) SEQ 2.54 2.06 ND ID NO 63 11(AMX200.B1) SEQ 4.01 3.01 ND ID NO 64 12 (AMX199.C2) SEQ 5.09 4.61 17 IDNO 65 13 (AMX200.B8) SEQ 4.13 3.13 ND ID NO 66 14 (AMX200.F11) SEQ 3.833.25 34 ID NO 67 15 (AMX200.D1) SEQ 1.26 1.06 ND ID NO 68 16 (AMX200.F9)SEQ 0.97 0.99 ND ID NO 69 17 (AMX199.B7) SEQ 4.08 3.65 29 ID NO 70 18(AMX200.D3) SEQ 2.68 2.41 36 ID NO 71 19 (AMX199.C4) SEQ 3.60 2.85 34 IDNO 72 20 (AMX200.E8) SEQ 1.04 1.03 ND ID NO 73 21 (AMX199.F10) SEQ 1.171.24 ND ID NO 74 22 (AMX199.F6) SEQ 1.37 1.30 ND ID NO 75 23 (AMX199.G5)SEQ 1.41 1.34 ND ID NO 76 24 (AMX199.F11) SEQ 1.44 1.35 ND ID NO 77 25(AMX199.H7) SEQ 1.32 1.14 ND ID NO 78 26 (AMX199.A10) 1.25 1.29 ND SEQID NO 79 27 (AMX199.G1) SEQ 1.19 1.26 ND ID NO 80 28 (AMX199.F1) SEQ1.32 1.36 ND ID NO 81 29 (AMX199.G4) SEQ 1.19 1.11 ND ID NO 82 30(AMX200.A11) 1.49 1.19 ND SEQ ID NO 83 31 DL.159.83.31 1.86 1.27 ND(AMX200.H8) SEQ ID NO 84 32 (AMX199.F8) SEQ 1.78 1.79 ND ID NO 85 33(AMX199.B6) SEQ 2.01 1.91 ND ID NO 86 34 (AMX199.D8) SEQ 1.89 2.00 ND IDNO 87 35 (AMX200.E10) SEQ 1.69 1.82 ND ID NO 88 36 (AMX202.H10) 1.921.76 ND SEQ ID NO 89 37 (AMX202.B8) SEQ 1.66 1.41 ND ID NO 90 38(AMX202.D6) SEQ 1.94 1.55 ND ID NO 91 39 (AMX202.A3) SEQ 2.28 2.10 ND IDNO 92 40 DL.159.83.98 1.27 1.27 ND (AMX202.A8) SEQ ID NO 93 41(AMX202.F6) SEQ 1.49 1.46 ND ID NO 94 *used human vWF A1 domain (SEQ IDNO 5) for aptamer screen and aptamer K_(D)s ND = not done

The nucleic acid sequences of the DNA aptamers characterized in Table 14above are described below, The unique sequence of each aptamer belowbegins at nucleotide 21, immediately following the sequenceCTACCTACGATCTGACTAGC (SEQ ID NO 52), and runs until it meets the 3′fixed nucleic acid sequence GCTTACTCTCATGTAGTTCC (SEQ ID NO 223).

Unless noted otherwise, individual sequences listed below arerepresented in the 5′ to 3′ orientation and were selected under DNASELEX™ wherein all of the nucleotides are deoxy.

DNA SELEX™ 1, Family #1

The predicted core nucleic acid binding sequence for DNA SELEX™ 1,Family #1 is shown in bold and underlined for aptamer AMX199.B3 (SEQ IDNO 54) and the consensus sequence (SEQ ID NO 95) below.

(AMX199.B3) SEQ ID NO 54 CTACCTACGATCTGACTAGCGGAATGAGAATGCTGATGGATTGCTCAGG TCTGCTGGCTGCTTACTCTCAT GTAGTTCC (AMX200.C11)SEQ ID NO 59 CTACCTACGATCTGACTAGCGGAATGAGAGTGCTGATGGATTGCTCAGGTCTGCTGGCTGCTTACTCTCATGTAGTTCC (AMX200.G11) SEQ ID NO 56CTACCTACGATCTGACTAGCGGAACGAGAATGCTGATGGATTGCTCAGGTCTGCTGGCTGCTTACTCTCATGTAGTTCC (AMX199.D11) SEQ ID NO 55CTACCTACGATCTGACTAGCGGAATGAGAATGCTGGTGGATTGCTCAGGTCTGCTGGCTGCTTACTCTCATGTAGTTCC (AMX200.D8) SEQ ID NO 58CTACCTACGATCTGACTAGCGGAATGAGAATGTTGATGGATTGCTCAGGTCTGCTGGCTGCTTACTCTCATGTAGTTCC (AMX200.D11) SEQ ID NO 57CTACCTACGATCTGACTAGCGGAATAAGAATGCTGATGGATTGCTCAGGTCTGCTGGCTGCTTACTCTCATGTAGTTCC (AMX199.F7) SEQ ID NO 61CTACCTACGATCTGACTAGCGGAATGAGAGTGCTGGTGGATTGCTCAGGTCTGCTGGCTGCTTACTCTCATGTAGTTCC (AMX200.A7) SEQ ID NO 62CTACCTACGATCTGACTAGCGGAATGAGAATGCTGATGGATTGTTCAGGTCTGCTGGCTGCTTACTCTCATGTAGTTCC (AMX202.D6) SEQ ID NO 91CTACCTACGATCTGACTAGCGGAATGAGAAGGCTGATGGATTGCTCAGGTCTGCTGGCTGCTTACTCTCATGTAGTTCC (AMX200.B1) SEQ ID NO 64CTACCTACGATCTGACTAGCGGAATGAGAATGCTGATGGATTGCCCAGGTCTGCTGGCTGCTTACTCTCATGTAGTTCC (AMX199.G10) SEQ ID NO 60CTACCTACGATCTGACTAGCGGAATGAGAATGTTGGTGGATTGCTCAGGTCTGCTGGCTGCTTACTCTCATGTAGTTCC (AMX200.B8) SEQ ID NO 66CTACCTACGATCTGACTAGCGGAATGAGAATGCTGATGGATTGCACAGGTCTGCTGGCTGCTTACTCTCATGTAGTTCC (AMX200.B9) SEQ ID NO 63CTACCTACGATCTGACTAGCGGAATGAGAATGCTGATGGATTGCTCAGGTCTGCTGACTGCTTACTCTCATGTAGTTCC (AMX202.B8) SEQ ID NO 90CTACCTACGATCTGACTAGCGGAATGAGTATGCTGATGGATTGCTCAGGTCTGCTGGCTGCTTACTCTCATGTAGTTCC (AMX202.H10) SEQ ID NO 89CTACCTACGATCTGACTAGCGGAATGAGAAGGCTGGTGGATTGCTCAGGTCTGCTGGCTGCTTACTCTCATGTAGTTCC (AMX200.F9) SEQ ID NO 69CTACCTACGATCTGACTAGCGGAATGAGGATGCTGATGGATTGGTCAGGTCTGCTGGCTGCTTACTCTCATGTAGTTCC (AMX202.A3) SEQ ID NO 92CTACCTACGATCTGACTAGCGGAATGAGAGCGCTGATGGATTGCTCAGGTCTGCTGGCTGCTTACTCTCATGTAGTTCC (AMX199.C2) SEQ ID NO 65CTACCTACGATCTGACTAGCGGAATGAGAATGCTGGTGGATTGCCCAGGTCTGCTGGCTGCTTACTCTCATGTAGTTCC (AMX200.F11) SEQ ID NO 67CTACCTACGATCTGACTAGCGGAATGAGGATGCTGGTGGATTGCTCAGGTCTGCTGGCTGCTTACTCTCATGTAGTTCC (AMX200.D1) SEQ ID NO 68CTACCTACGATCTGACTAGCGGAATGAGAGTGCTGATGGATTGCTCAGGTCTACTGGCTGCTTACTCTCATGTAGTTCC (AMX199.C4) SEQ ID NO 72CTACCTACGATCTGACTAGCGGAATGAGGATGCTGATGGATTGCACAGGTCTGCTGGCTGCTTACTCTCATGTAGTTCC (AMX200.D3) SEQ ID NO 71CTACCTACGATCTGACTAGCGCAATGAGGATGCTGATGGATTGCTCAGGTCTGCTGGCTGCTTACTCTCATGTAGTTCC (AMX199.B7) SEQ ID NO 70CTACCTACGATCTGACTAGCGGGATGAGAGTGCTGGTGGATTGCTCAGGTCTGCTGGCTGCTTACTCTCATGTAGTTCC (AMX200.E8) SEQ ID NO 73CTACCTACGATCTGACTAGCGGAATGAGGATGCTGGTGGATTGCTCAGGTCTGTTGGCTGCTTACTCTCATGTAGTTCC

The Consensus sequence for DNA SELEX™ 1, Family #1 is as follows:

SEQ ID NO 95 CTACCTACGATCTGACTAGCGGA ATGAGRATGYTGRTGGATTGCHAGGTCTRYTGRCTGCTTACTCTCAT GTAGTTCCWhere Y=C or T, R=A or G and H=A, C or TDNA SELEX™ 1 Family #2

(AMX199.F10) SEQ ID NO 74CTACCTACGATCTGACTAGCGAAACACTAGGTTGGTTAGGATTGGTGTGTTTCCGTTCTGCTTACTCTCATGTAGTTCC AMX199.F6) SEQ ID NO 75CTACCTACGATCTGACTAGCGAAACACTAGGTTGGTTAGGATTGGTGTGTTCCCGCTCTGCTTACTCTCATGTAGTTCC (AMX199.H7) SEQ ID NO 78CTACCTACGATCTGACTAGCGAAACACTAGGTTGGTTAGGATTGGTGTGTTTCCGCTTTGCTTACTCTCATGTAGTTCC (AMX199.G5) SEQ ID NO 76CTACCTACGATCTGACTAGCGAAACACTAGGTTGGTTAGGATTGGTGTGTTCCCGCCCTGCTTACTCTCATGTAGTTCC (AMX199.F11) SEQ ID NO 77CTACCTACGATCTGACTAGCGAAACACTAGGTTGGTTAGGATTGGTGTGTTTCTGCTCTGCTTACTCTCATGTAGTTCC (AMX199.A10) SEQ ID NO 79CTACCTACGATCTGACTAGCGGAACACTAGGTTGGTTAGGATTGGTGTGTTCCCGTTTTGCTTACTCTCATGTAGTTCC (AMX199.G1) SEQ ID NO 80CTACCTACGATCTGACTAGCGAAACACTAGGTTGGTTAGGATTGGTGTGTTCCCGCTTTGCTTACTCTCATGTAGTTCC (AMX199.G4) SEQ ID NO 82CTACCTACGATCTGACTAGCGAAACACTAGGTTGGTTAGGGTTGGTGTGTTCCCGCTTTGCTTACTCTCATGTAGTTCC (AMX199.F1) SEQ ID NO 81CTACCTACGATCTGACTAGCGAAACACTAGGTTGGTTAGGATTGGTGTGTTCCCGCTATGCTTACTCTCATGTAGTTCCThe consensus sequence for DNA SELEX™ Family #2 is as follows:

SEQ ID NO 96 CTACCTACGATCTGACTAGCGRAACACTAGGTTGGTTAGGRTTGGTGTGTTYCYGYYHGCTTACTCTCATGTAGTTCC

Where Y=C or T, R=A or G and H=A, C or T

DNA SELEX™ 1 Family #3

(AMX199.B6) SEQ ID NO 86CTACCTACGATCTGACTAGCAAGGGGATTGGCTCCGGGTCTGGCGTGCTTGGTACCTCCGGCTTACTCTCATGTAGTTCC (AMX199.D8) SEQ ID NO 87CTACCTACGATCTGACTAGCAAGGGGATTGGCTCCGGGTCTGGCGTGCTTGGCATCTTCGGCTTACTCTCATGTAGTTCC (AMX199.F8) SEQ ID NO 85CTACCTACGATCTGACTAGCAAGGGGATTGGCTCCGGGTCTGGCGTGCTCGGCACCTTTGGCTTACTCTCATGTAGTTCC (AMX200.E10) SEQ ID NO 88CTACCTACGATCTGACTAGCAAGGGGATTGGCTCCGGGTCTGGCGTGCTCGGCACCTTTGGCTTACTCTCATGTAGTTCC (AMX202.F6) SEQ ID NO 94CTACCTACGATCTGACTAGCAAGGGGATTGGCTCCGGGTCTGGCGTGCTCGGCACCTTCGGCTTACTCTCATGTAGTTCC (AMX202.A8) SEQ ID NO 93CTACCTACGATCTGACTAGCAAGGGGATTGGCTCCGGGTCTGGCGTGCTCGGCACTTCCGGCTTACTCTCATGTAGTTCC DNA SELEX™ 1, Family #4 (AMX200.A11) SEQID NO 83 CTACCTACGATCTGACTAGCTGAGTAGTTAGTAACTTTTTATTATGGTTTGGTGGGTCTGGCTTACTCTCATGTAGTTCC (AMX200.H8) SEQ ID NO 84CTACCTACGATCTGACTAGCTGAGTAGTCAGTAATTTTTTATTATGGTTTGGTGGGCCTGGCTTACTCTCATGTAGTTCC

Example 1D Selection #2 of DNA vWF Aptamers

A single set of DNA selections were done using full length human vWF andrabbit vWF domain A1 in a cross selection. While not wishing to be boundby any theory, our hypothesis is that such a selection should requiresuccessfully selected aptamers to bind to full length vWF, to bind tothe A1 domain specifically and to cross react between human and rabbitproteins. The dominant sequence family from this second set of DNAselections binds to full length human vWF, rabbit vWF domain A1 and isfunctional in both the FACS and BIPA biological assays as described inExample 3 below.

Selections were performed to identify aptamers that bind to full lengthhuman vWF and rabbit vWF A1 domain, using a full length human vWF/rabbitvWF A1 domain alternating selection. This selection used a nucleotidepool consisting of deoxy-nucleotides (DNA). The selection strategyyielded high affinity aptamers specific for full length human vWF andrabbit vWF A1 domain which had been immobilized on a hydrophobic plate.

Pool Preparation

A DNA template with the sequence5′-CTACCTACGATCTGACTAGCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGCTTACTCTCATGTAGTTCC-3′(SEQ ID NO 51) (ARC 493) was synthesized using an ABI EXPEDITE™ DNAsynthesizer, and deprotected by standard methods. The series of N's inthe DNA template (SEQ ID NO 51) can be any combination of nucleotidesand gives rise to the unique sequence region of the resulting aptamers.The template was PCR amplified with the primers(5′-CTACCTACGATCTGACTAGC-3′) (SEQ ID NO 52) and(5′-AGGAACTACATGAGAGTAAGC(OH)-3′) (SEQ ID NO 53 ) under standardconditions. The PCR product was subjected to alkaline hydrolysis (333 mMNaOH, 90° C., 15 min) followed by precipitation. The strands wereseparated on a 10% denaturing polyacrylamide gel and the single strandedDNA pool, which migrated with a lower mobility, was excised from thegel, passively eluted, and precipitated with isopropanol.

Selection

For the first three rounds of the full length human vWF/rabbit vWF A1domain alternating selection, 24 pmoles of full length human vWF (SEQ IDNO 7) and 24 moles of rabbit vWF A1 domain (SEQ ID NO 6) wereimmobilized. In the subsequent rounds, the protein target was alternatedeach round between full length human vWF and rabbit vWF A1 domain. Inall cases, after one hour of protein immobilization, the supernatant wasremoved and the wells were washed 4 times with 120 μL 1× Dulbecco's PBS.The protein-immobilized well was then blocked with 100 uL blockingbuffer (1× Dulbecco's PBS with 1% BSA) for 1 hour at room temperature.In Round one, 333 pmoles of pool DNA (2×10¹⁴ unique molecules) wereincubated in 100 μL 1× Dulbecco's PBS in the wells containingBSA-blocked immobilized protein target for 1 hour. The supernatant wasthen removed and the wells were washed 4 times with 120 μL 1× Dulbecco'sPBS. In later rounds, additional washes were added to increase thestringency of the positive selection step (see Table 15). At Round 8,the selection was split to include a high salt wash condition as apossible means to increase the stringency of the SELEX™ (using 1×Dulbecco's PBS+400 mM NaCl) (see Table 15). Starting at Round 2 and inall subsequent rounds, two negative selection steps were included beforethe positive selection step. First, the pool DNA was incubated for 1hour at room temperature in an unblocked well to remove any plasticbinding sequences from the pool. In the second negative selection step,the DNA was transferred to a BSA blocked well (not containing theprotein target) for 1 hour at room temperature to remove any BSA bindingsequences from the pool prior to the positive selection. Starting atRound 2 and in all subsequent rounds, 0.1 mg/mL tRNA and 0.1 mg/mLsalmon sperm DNA were spiked into the positive selection reaction asnon-specific competitors.

In all cases, the pool DNA bound to the immobilized protein target waseluted with 2×100 μL washes with elution buffer (preheated to 90° C., 7M Urea, 100 mM NaOAc pH 5.3, 3 mM EDTA) for five minutes. Both elutionswere pooled and precipitated by the addition of ethanol, then amplifiedin an initial PCR reaction (100 μL reactions including the 5′-primeraccording to SEQ ID NO 52, the 3′-primer according to SEQ ID NO 53, andTaq polymerase, (New England BioLabs, Cat.# M0267L, Beverly, Mass.). PCRreactions were done under the following conditions: a) denaturationstep: 94° C. for 2 minutes; b) cycling steps: 94° C. for 30 seconds, 52°C. for 30 seconds, 72° C. for 1 minute; c) final extension step: 72° C.for 3 minutes. The cycles were repeated until sufficient PCR product wasgenerated. The minimum number of cycles required to generate sufficientPCR product is reported in Table 15 as the “PCR Threshold”. 10 μL of thePCR product was added to another 300 μL of PCR mix for a prep-scale PCRreaction. The prep-scale PCR product was ethanol precipitated and wassubjected to alkaline hydrolysis (333 mM NaOH, 90° C., 15 min). Thestrands were separated on a 10% denaturing polyacrylamide gel and thesingle stranded DNA pool, which migrated with a lower mobility, wasexcised from the gel, passively eluted, and precipitated withisopropanol. In all cases, an equivalent of half of the total singlestranded DNA product was carried forward as the starting pool for thesubsequent round of selection.

TABLE 15 Full length human vWF/rabbit vWF A1 domain alternatingselection conditions using a DNA pool Round Target Washes PCR ThresholdPurification 1 24 pmol full length 4 × 120 uL 15 Gel purify human/ 24pmol rA1 2 24 pmol full length 4 × 120 uL 13 Gel purify human/ 24 pmolrA1 3 24 pmol full length 4 × 120 uL 10 Gel purify human/ 24 pmol rA1 424 pmol full length 4 × 120 uL 10 Gel purify human vWF 5 24 pmol rA1 4 ×120 uL 10 Gel purify 6 24 pmol full length 8 × 120 uL 10 Gel purifyhuman vWF 7 24 pmol rA1 8 × 120 uL 10 Gel purify Normal High Salt NormalHigh Salt Wash Wash Wash Wash 8 24 pmol full length 8 × 120 uL 8 × 120uL 10 10 Gel purify human vWF 9 24 pmol rA1 8 × 120 uL 8 × 120 uL 10 13Gel purify 10  24 pmol full length 8 × 120 uL 8 × 120 uL 10 10 Gelpurify human vWF 11  24 pmol rA1 8 × 120 uL 8 × 120 uL 10 10 Gel purifyvWF Binding Analysis

The selection progress was monitored using a sandwich filter bindingassay. The 5′-³²P-labeled pool DNA (trace concentration) was incubatedwith either a no target protein control, 100 nM full length human vWF(Calbiochem Cat.#681300, La Jolla, Calif.), or 100 nM rabbit vWF A1domain, in 1× Dulbecco's PBS containing 0.1 mg/mL tRNA, and 0.1 mg/mLsalmon sperm DNA, and 0.1 mg/mL BSA in a (final volume of 50 uL) for 30minutes at room temperature and then applied to a nitrocellulose andnylon filter sandwich in a dot blot apparatus (Schleicher and Schuell,Keene, N.H.). The percentage of pool DNA bound to the nitrocellulose wascalculated after Rounds 7 and 9 by screening with a no protein targetcontrol, 30 nM /100 nM full length human vWF (Calbiochem Cat.#681300, LaJolla, Calif.), and 30 nM/100 nM rabbit vWF A1 domain (SEQ ID NO 6).Pool binding was compared to that of the naïve pool DNA (Round 0). Theresults of the DNA pool binding analyses are found in Table 16 below.

TABLE 16 full length human vWF/rabbit vWF A1 domain DNA selection poolbinding assays. full length rabbit vWF Pool human vWF A1 domain NoSelection Round 30 nM 100 nM 30 nM 100 nM Protein Naïve Pool Round 040.3% 39.8% 41.6% 45.5% 35.2% Human Round 7 59.4% 66.9% 58.9% 68.3%39.9% vWF/rA1 Naïve Pool Round 0 53.0% 55.1% 53.9% 56.9% 52.6% HumanRound 9 70.9% 65.0% 71.7% 81.6% 54.5% vWF/rA1 Human Round 9 72.1% 73.8%74.5% 82.3% 59.7% vWF/rA1 High Salt Wash

When a significant positive ratio of binding of DNA in the presence ofhuman or rabbit vWF A1 domain versus in the absence of protein was seen,the pools were cloned using the TOPO TA cloning kit (InvitrogenCat.#45-0641, Carlsbad, Calif.) according to the manufacturer'sinstructions. Round 7 and 11 pool templates were cloned and sequenced(218 total sequences), producing 146 unique clones within 12 sequencefamilies of which sequences from six families show vWF target bindingactivity. All unique clones were assayed twice in a 3-point dot blotscreen (no protein target control, 20 nM full length human vWF(Calbiochem Cat.#681300, La Jolla, Calif.), or 20 nM rabbit vWF A1domain. The data is presented in the third and fourth columns of Table17 below as the ratio of the fraction of the aptamer bound to thenitrocellulose in the presence of the target protein to the fraction ofaptamer bound in the absence of the target protein.

Based on this initial screen, K_(D)s were determined for 3 of the vWFdependent binding sequences using the dot blot assay. For K_(D)determination, aptamers were 5′end labeled with γ-³²P ATP and weretested for direct binding to full length human vWF and rabbit vWF A1domain. A 12 point protein titration was used in the dot blot assay (300nM, 100 nM, 30 nM, 10 nM, 3 nM, 1 nM, 300 pM, 100 pM, 30 pM, 10 pM, 3pM, 0 pM) in 1× DPBS plus 0.1 mg/mL BSA at room temperature for 30minutes. K_(D) values were calculated by fitting the equationy=(max/(1+K/protein))+yint using KaleidaGraph (KaleidaGraph v. 3.51,Synergy Software). Results of protein binding characterization aretabulated in Table 17 below.

TABLE 17 Full length human vWF and rabbit vWF A1 domain DNA aptamerbinding activity* Screen full length rabbit Screen- Rabbit/ human vWF A1Human/No No vWF K_(D) domain K_(D) # Aptamer Protein Protein (nM) (nM) 1AMX237.A11 2.66 2.38 ND ND (SEQ ID NO 98) 2 AMX237.A2 2.60 2.34 ND ND(SEQ ID NO 99) 3 AMX238.D12 2.40 2.19 ND ND (SEQ ID NO 100) 4 AMX237.H51.63 1.61 ND ND (SEQ ID NO 101) 5 AMX237.E2 1.61 1.61 ND ND (SEQ ID NO102) 6 AMX237.B4 1.42 1.38 ND ND (SEQ ID NO 103) 7 AMX237.E9 2.13 2.06ND ND (SEQ ID NO 104) 8 AMX237.D11 1.16 1.17 ND ND (SEQ ID NO 105) 9AMX238.G5 3.06 2.68 ND ND (SEQ ID NO 106) 10 AMX237.C7 1.15 1.16 ND ND(SEQ ID NO 107) 11 AMX238.D5 1.46 1.40 ND ND (SEQ ID NO 108) 12AMX237.B11 2.87 2.57 ND ND (SEQ ID NO 109) 13 AMX237.F6 1.20 1.21 ND ND(SEQ ID NO 110) 14 AMX238.D8 2.02 1.97 ND ND (SEQ ID NO 111) 15AMX238.G6 1.25 1.22 ND ND (SEQ ID NO 112) 16 AMX236.F8 1.14 1.13 ND ND(SEQ ID NO 113) 17 AMX237.G6 3.80 3.63 0.20 47 (SEQ ID NO 114) 18AMX238.E9 3.44 3.36 0.39 5.3 (SEQ ID NO 115) 19 AMX238.E7 3.02 2.83 NDND (SEQ ID NO 116) 20 AMX238.F3 2.83 2.72 ND ND (SEQ ID NO 117) 21AMX238.H5 3.75 3.46 0.33 6.0 (SEQ ID NO 118) 22 AMX237.C11 2.04 1.95 NDND (SEQ ID NO 119) 23 AMX238.F2 2.84 2.76 ND ND (SEQ ID NO 120) 24AMX237.F9 2.21 2.31 ND ND (SEQ ID NO 121) 25 AMX237.F12 1.95 2.08 ND ND(SEQ ID NO 122) 26 AMX237.C9 2.05 2.19 ND ND (SEQ ID NO 123) 27AMX237.F10 2.90 2.90 ND ND (SEQ ID NO 124) 28 AMX236.H2 2.12 2.06 ND ND(SEQ ID NO 125) 29 AMX237.C5 2.55 2.36 ND ND (SEQ ID NO 126) 30AMX236.A12 2.64 2.41 ND ND (SEQ ID NO 127) 31 AMX236.B8 1.66 1.88 ND ND(SEQ ID NO 128) 32 AMX236.A11 2.02 2.02 ND ND (SEQ ID NO 129) 33AMX237.D5 1.41 1.48 ND ND (SEQ ID NO 130) 34 AMX236.E6 1.31 1.49 ND ND(SEQ ID NO 131) 35 AMX236.C12 1.99 2.24 ND ND (SEQ ID NO 132) 36AMX237.H10 1.71 1.94 ND ND (SEQ ID NO 133) 37 AMX237.G7 2.68 2.54 ND ND(SEQ ID NO 134) 38 AMX237.H8 1.21 1.41 ND ND (SEQ ID NO 135) 39AMX236.G4 1.70 1.72 ND ND (SEQ ID NO 136) 40 AMX236.C1 1.03 3.28 ND ND(SEQ ID NO 137) 41 AMX237.E10 1.12 6.04 ND ND (SEQ ID NO 138) 42AMX238.F5 1.05 4.40 ND ND (SEQ ID NO 139) 43 AMX237.C1 0.76 3.47 ND ND(SEQ ID NO 140) 44 AMX237.B12 1.13 4.67 ND ND (SEQ ID NO 141) 45AMX238.A6 0.92 3.47 ND ND (SEQ ID NO 142) 46 AMX238.A11 0.85 4.54 ND ND(SEQ ID NO 143) 47 AMX236.C6 1.06 5.77 ND ND (SEQ ID NO 144) 48AMX238.F6 1.18 5.36 ND ND (SEQ ID NO 145) 49 AMX236.E2 0.93 3.59 ND ND(SEQ ID NO 146) 50 AMX238.G4 1.09 1.39 ND ND (SEQ ID NO 147) 51AMX238.H9 1.11 1.32 ND ND (SEQ ID NO 148) 52 AMX237.B1 2.00 2.10 ND ND(SEQ ID NO 149) 53 AMX238.A3 1.36 1.03 ND ND (SEQ ID NO 150) 54AMX237.C4 0.97 1.31 ND ND (SEQ ID NO 151) 55 AMX237.E5 0.97 1.15 ND ND(SEQ ID NO 152) 56 AMX237.F1 0.98 1.22 ND ND (SEQ ID NO 153) 57AMX237.F5 0.99 1.22 ND ND (SEQ ID NO 154) 58 AMX238.H11 0.98 1.14 ND ND(SEQ ID NO 155) 59 AMX237.G2 1.02 1.16 ND ND (SEQ ID NO 156) 60AMX238.A12 1.23 0.99 ND ND (SEQ ID NO 157) 61 AMX236.C9 1.24 1.00 ND ND(SEQ ID NO 158) 62 AMX236.H1 1.10 1.14 ND ND (SEQ ID NO 159) 63AMX236.F7 1.18 1.20 ND ND (SEQ ID NO 160) 64 AMX236.B3 1.54 1.41 ND ND(SEQ ID NO 161) 65 AMX238.D9 1.22 0.97 ND ND (SEQ ID NO 162) 66AMX238.F7 1.20 1.88 ND ND (SEQ ID NO 163) 67 AMX236.G1 1.47 1.51 ND ND(SEQ ID NO 164) *used full length human vWF (SEQ ID NO 7) and rabbit vWFA1 domain (SEQ ID NO 6) for aptamer screen and aptamer K_(D)s ND = notdone

The nucleic acid sequences of the DNA aptamers characterized in Table 17above are described below. The unique sequence of each aptamer belowbegins at nucleotide 21, immediately following the sequenceCTACCTACGATCTGACTAGC (SEQ ID NO 52), and runs until it meets the 3′fixed nucleic acid sequence GCTTACTCTCATGTAGTTCC (SEQ ID NO 223).

Unless noted otherwise, individual sequences listed below arerepresented in the 5′ to 3′ orientation and were selected under DNASELEX™ wherein all of the nucleotides are deoxy.

vWF DNA SELEX™ 2, Family 1.1

Families 1.1 and 1.2 yielded the parent of ARC1029 (SEQ ID NO 214). Thepredicted core nucleic acid binding sequences to the target vonWillebrand Factor are underlined and shown in bold for aptamersAMX237.E9 (SEQ ID NO 104) and AMX238.H5 (SEQ ID NO 118) below.

AMX237.E9 (SEQ ID NO 104) CTACCTACGATCTGACTAGCTCCAGTGTTTTGTCTAATAACCGTGCGGTG CCTCCGTGAGCT TACTCTCATGTAGTTCCAMX237.B11 (SEQ ID NO 109)CTACCTACGATCTGACTAGCTCCAGTGTTTTATCTAATAACCGTGCGGTGCCTCCGTGAGCTTACTCTCATGTAGTTCC AMX237.A11 (SEQ ID NO 98)CTACCTACGATCTGACTAGCTCCAGTGTTTTATCCAATAACCGTGCGGTGCCTCCGTGAGCTTACTCTCATGTAGTTCC AMX238.G5 (SEQ ID NO 106)CTACCTACGATCTGACTAGCTCCAGTGTTTTATCCAACAACCGTGCGGTGCCTCCGTGAGCTTACTCTCATGTAGTTCC AMX238.D8 (SEQ ID NO 111)CTACCTACGATCTGACTAGCTCCAGTGTTTTATCCAACAACCGTGCGGTGCCTCCGTGAGCTTACTCTCATGTAGTTCC AMX237.E2 (SEQ ID NO 102)CTACCTACGATCTGACTAGCTCCAGTGTTTCATCTAATAACCGTGCGGTGCCTCCGTGAGCTTACTCTCATGTAGTTCC AMX237.H5 (SEQ ID NO 101)CTACCTACGATCTGACTAGCTCCAGTGTTTCATTTAATAACCGTGCGGTGCCTCCGTGAGCTTACTCTCATGTAGTTCC AMX238.D5 (SEQ ID NO 108)CTACCTACGATCTGACTAGCTCCAGTGTTTTATTCAATAACCGTGCGGTGTCTCCGTGAGCTTACTCTCATGTAGTTCC AMX237.A2 (SEQ ID NO 99)CTACCTACGATCTGACTAGCTCCAGTGTTTCATCCAATAACCGTGCGGTGCCTCCGTGAGCTTACTCTCATGTAGTTCC AMX238.D12 (SEQ ID NO 100)CTACCTACGATCTGACTAGCTCCAGTGTTTCATTCAATAACCGTGCGGTGCCTCCGTGAGCTTACTCTCATGTAGTTCC AMX237.F6 (SEQ ID NO 110)CTACCTACGATCTGACTAGCTCCAGTGTTTTATCTAATAACGTGCGGTGCCTCCGTGATGCTTACTCTCATGTAGTTCC AMX237.D11 (SEQ ID NO 105)CTACCTACGATCTGACTAGCTCCAGTGTTTTATATAATAACCGTGCGGTGCCTCCGTGATGCTTACTCTCATGTAGTTCC AMX237.B4 (SEQ ID NO 103)CTACCTACGATCTGACTAGCTCCAGTGTTTCATCCAATAACCGTGCGGTGCTTCCGTGAGCTTACTCTCATGTAGTTCC AMX236.F8 (SEQ ID NO 113)CTACCTACGATCTGACTAGCTCCAGTGTTTTATCCAATAACCGTGCGGTGCCTCCGTGATGCTTACTCTCATGTAGTTCC AMX237.C7 (SEQ ID NO 107)CTACCTACGATCTGACTAGCTCCAGTGTTTTATTCAATAACCGTGCGGTGCCTCCGTGATGCTTACTCTCATGTAGTTCC AMX238.G6 (SEQ ID NO 112)CTACCTACGATCTGACTAGCTCCAGTGTTTTATCCAATAACCGTGCGGGGCCTCCGTGATGCTTACTCTCATGTAGTTCC vWF DNA SELEX™ 2, Family # 1.2 AMX238.H5(SEQ ID NO 118) CTACCTACGATCTGACT AGCGTGCAGTGCCTATTCTAGGCCGTGCGGTGCCTCCGTCACGCT TACTCTCATGTAGTTCC AMX237.C11 (SEQ ID NO 119)CTACCTACGATCTGACTAGCGTGCAGTGCCTATTCTAGGCCGTGCGGTGCCTCCGTCATGCTTACTCTCATGTAGTTCC AMX238.E7 (SEQ ID NO 116)CTACCTACGATCTGACTAGCGTGCAGTGCCTATTTTAGGCCGTGCGGTGCCTCCGTCACGCTTACTCTCATGTAGTTCC AMX237.G6 (SEQ ID NO 114)CTACCTACGATCTGACTAGCGTGCAGTGCCTATTCCAGGCCGTGCGGTGCCTCCGTCACGCTTACTCTCATGTAGTTCC AMX238.F2 (SEQ ID NO 120)CTACCTACGATCTGACTAGCATGCAGTGCCCATTCTAGGCCGTGCGGTGCCTCCGTCATGCTTACTCTCATGTAGTTCC AMX238.E9 (SEQ ID NO 115)CTACCTACGATCTGACTAGCGTGCAGTGCCCATCTTAGGCCGTGCGGTGCCTCCGTCACGCTTACTCTCATGTAGTTCC AMX238.F3 (SEQ ID NO 117)CTACCTACGATCTGACTAGCGTGCAGTGCCTATTTTAGGTCGTGCGGGGCCTCCGTCACGCTTACTCTCATGTAGTTCC AMX237.F10 (SEQ ID NO 124)CTACCTACGATCTGACTAGCGTGCAGTGCCCATTCCAGGCCGTGCGGTATCCTCCGTCACGCTTACTCTCATGTAGTTCC AMX237.C5 (SEQ ID NO 126)CTACCTACGATCTGACTAGCGTGCAGTGCCTATCTCAGGCCGTGCGGTATCCTCCGTCACGCTTACTCTCATGTAGTTCC AMX236.H2 (SEQ ID NO 125)CTACCTACGATCTGACTAGCGTGCAGTGCCTATCCCAGGCCGTGCGGTAGCCTCCGTCACGCTTACTCTCATGTAGTTCC

The predicted secondary structure and core nucleic acid sequencesrequired for binding to the vWF target of some embodiments of theinvention comprised in Family#1 of this aptamer selection is depicted inFIG. 15 as SEQ ID NO 220.

vWF DNA SELEX™ 2. Binding Family #2

AMX237.C9 (SEQ ID NO 123)CTACCTACGATCTGACTAGCTTGGTAGTGACTTTGTGGAGCTGCGGTTTGGTCGACGTCAGCTTACTCTCATGTAGTTCC AMX237.F12 (SEQ ID NO 122)CTACCTACGATCTGACTAGCTTGGTAGCGATTTTGTGGAGCTGCGGTTTGGTCGACGTCAGCTTACTCTCATGTAGTTCC AMX237.F9 (SEQ ID NO 121)CTACCTACGATCTGACTAGCTTGGTAGCGATTCTGTGGAGCTGCGGTTTGGTCGACGTCAGCTTACTCTCATGTAGTTCC AMX237.G7 (SEQ ID NO 134)CTACCTACGATCTGACTAGCTTGGTAGCGACTTTGTGGAGCTGCGGTTTGGTCGACGTCAGCTTACTCTCATGTAGTTCC AMX236.A12 (SEQ ID NO 127)CTACCTACGATCTGACTAGCTTGGTAGCGACTCTGTGGAGCTGCGGTTTGGTCGACGTCAGCTTACTCTCATGTAGTTCC AMX236.G4 (SEQ ID NO 136)CTACCTACGATCTGACTAGCTTGGTAGCGACTTTGTGGAGATGCGGTTTGGTTGACGTCAGCTTACTCTCATGTAGTTCC AMX236.C12 (SEQ ID NO 132)CTACCTACGATCTGACTAGCTTGGTAGCGACTCCGTGGAGCTGCGGTTTGGTCGACGTCAGCTTACTCTCATGTAGTTCC AMX236.A11 (SEQ ID NO 129)CTACCTACGATCTGACTAGCTTGGTAGCGACTCTGTGGAGCTGCGGTCTGGCCGACGTCAGCTTACTCTCATGTAGTTCC AMX236.E6 (SEQ ID NO 131)CTACCTACGATCTGACTAGCTTGGTAGCGACCCTGTGGAGCTGCGGTTTGGTCGACGTCAGCTTACTCTCATGTAGTTCC AMX236.B8 (SEQ ID NO 128)CTACCTACGATCTGACTAGCTTGGTAGCGACTCTGTGGAGCTGCGGTCTGGTCGACGTCAGCTTACTCTCATGTAGTTCC AMX237.H8 (SEQ ID NO 135)CTACCTACGATCTGACTAGCTTGGTAGCGACTTTGTGGAGCTGCGGTTTGGTCGACATCAGCTTACTCTCATGTAGTTCC AMX237.D5 (SEQ ID NO 130)CTACCTACGATCTGACTAGCTTGGTAGCGACACTGTGGAGCTGCGGTTTGGTTGACGTCAGCTTACTCTCATGTAGTTCC AMX237.H10 (SEQ ID NO 133)CTACCTACGATCTGACTAGCTTGGTAGCGACTCAGAGGAGCTGCGGTTTGGTCGACGTCAGCTTACTCTCATGTAGTTCC AMX237.B1 (SEQ ID NO 149)CTACCTACGATCTGACTAGCTTGGTAGCGACACAGTGGAGCTGCGGTTTGGTCGACGTCAGCTTACTCTCATGTAGTTCCThe consensus sequence for DNA SELEX™ 2 Family #2 is as follows:

SEQ ID NO 325 CTACCTACGATCTGACTAGCTTGGTAG Y GA Y (Y/A) Y (Y/A) G (T/A)GGAG (C/A) TGCGGT Y TGG YY GAC R TCAGCTTACTC TCATGTAGTTCCWhere Y=C or T, R=A or G and (Y/A)=C, T or A,vWF DNA SELEX™ 2, Binding Family #3

This family is equivalent to vWF DNA SELEX™ 1, Family #1 described abovein that the sequences in both families are more than 90% identical.

AMX238.A11 (SEQ ID NO 143)CTACCTACGATCTGACTAGCGGAATGAGAGTGTTGGTGGATTGCTCAGGTCTGCTGGCTGCTTACTCTCATGTAGTTCC AMX237.C1 (SEQ ID NO 140)CTACCTACGATCTGACTAGCGGAATGAGGATGCTGGTGGATTGCTCAGGTCTGCTGGCTGCTTACTCTCATGTAGTTCC AMX236.C6 (SEQ ID NO 144)CTACCTACGATCTGACTAGCGGAATGAGAGTGCTGGTGGATTGCTCAGGTCTGCTGGCTGCTTACTCTCATGTAGTTCC AMX236.C1 (SEQ ID NO 137)CTACCTACGATCTGACTAGCGGAATGAGAATGTTGGTGGATTGCTCAGGTCTGCTGGCTGCTTACTCTCATGTAGTTCC AMX237.E10 (SEQ ID NO 138)CTACCTACGATCTGACTAGCGGAATGAGAATGCTGGTGGATTGCTCAGGTCTGCTGGCTGCTTACTCTCATGTAGTTCC AMX238.F6 (SEQ ID NO 145)CTACCTACGATCTGACTAGCGGAATGAGTATGCTGGTGGATTGCTCAGGTCTGCTGGCTGCTTACTCTCATGTAGTTCC AMX236.E2 (SEQ ID NO 146)CTACCTACGATCTGACTAGCGGAATGAGTATGCTGATGGATTGCTCAGGTCTGCTGGCTGCTTACTCTCATGTAGTTCC AMX237.B12 (SEQ ID NO 141)CTACCTACGATCTGACTAGCGGAATGAGAATGCAGGTGGATTGCTCAGGTCTGCTGGCTGCTTACTCTCATGTAGTTCC AMX238.A6 (SEQ ID NO 142)CTACCTACGATCTGACTAGCGGAATGAGAATGCAGATGGATTGCTCAGGTCTGCTGGCTGCTTACTCTCATGTAGTTCC AMX238.F5 (SEQ ID NO 139)CTACCTACGATCTGACTAGCGGAATGAGAAGCTGGTGGATTGCTCAGGTCTGCTGGCTGCTTACTCTCATGTAGTTCCvWF DNA SELEX™ 2, Binding Family #4

AMX237.G2 (SEQ ID NO 156)CTACCTACGATCTGACTAGCTTTCAGTCTTTCATATTTATAGGGTTTGGCATTGGGTCTGGCTTACTCTCATGTAGTTCC AMX237.C4 (SEQ ID NO 151)CTACCTACGATCTGACTAGCTTTCAGTCTTCCACATTTATAGGGTTTGGCATTGGGTCTGGCTTACTCTCATGTAGTTCC AMX237.F5 (SEQ ID NO 154)CTACCTACGATCTGACTAGCTTTTAGTCTTCCACATTTATAGGGTTTGGCATTGGGTCTGGCTTACTCTCATGTAGTTCC AMX238.H11 (SEQ ID NO 155)CTACCTACGATCTGACTAGCTTTCAGTCTTTCACATTTATAGGGTTTGGCATTGGGTCTGGCTTACTCTCATGTAGTTCC AMX238.G4 (SEQ ID NO 147)CTACCTACGATCTGACTAGCTTGTCGCACTTTTGGTTGGTCTGGTTGGTTCTAAGTGCGCTTACTCTCATGTAGTTCC AMX237.E5 (SEQ ID NO 152)CTACCTACGATCTGACTAGCTTTCAGTCTTCTACATTTATAGGGTTTGGCATTGGGTCTGGCTTACTCTCATGTAGTTCC AMX238.H9 (SEQ ID NO 148)CTACCTACGATCTGACTAGCTTGTCGCACTTTTGGTTGGTCTGGTTGGTTTTAAGTGCGCTTACTCTCATGTAGTTCC AMX237.F1 (SEQ ID NO 153)CTACCTACGATCTGACTAGCTTTCAGTCTTCCACGTTTATAGGGTTTGGCATTGGGTCTGGTTACTCTCATGTAGTTCCvWF DNA SELEX™ 2, Family #5

AMX236.G1 (SEQ ID NO 164)CTACCTACGATCTGACTAGCCTCAGATTGACTCCGGCTGACTTGTTTTAATCTTCTGAGTGCTTACTCTCATGTAGTTCC AMX236.B3 (SEQ ID NO 161)CTACCTACGATCTGACTAGCCTTACCTATTCCCTTCTGCGGAATACGTCGAGTACTATGCTTACTCTCATGTAGTTCC AMX236.F7 (SEQ ID NO 160)CTACCTACGATCTGACTAGCCCCCACTTATCGTGTACCTTATGATATGTCGAATACTCTTGCTTACTCTCATGTAGTTCC AMX236.H1 (SEQ ID NO 159)CTACCTACGATCTGACTAGCCTCAGATTGACTCCGGCCGACTTGTTTTAATCTTCTGAGTGCTTACTCTCATGTAGTTCCThe consensus sequences for DNA SELEX™ 2 Family #5 are as follows:

SEQ ID NO 326

Family 5.1=SEQ ID NO 164 and SEQ ID NO 159

CTACCTACGATCTGACTAGCCTCAGATTGACTCCGGCYGACTTGTTTTAATCTTCTGAGTGCTTACTCTCATGTAGTTCCWherein Y=C or T

SEQ ID NO 327

Family 5.2=SEQ ID NO 161 & SEQ ID NO 160

CTACCTACGATCTGACTAGCC YY AC Y TAT Y (C/G) Y (C/G) T (T/A) C Y (G/T) Y R(G/T) R ATA Y GTCGA R TACT (A/C) TGCTTACTCTCATGTAGTrCCWhere Y=C or T, R=A or GvWF DNA SELEX™ 2, Family #6

AMX238.A3 (SEQ ID NO 150)CTACCTACGATCTGACTAGCTCAAAGTATTACTTATTGGCAATAAGTCGTTTACTCTATAGCTTACTCTCATGTAGTTCC AMX238.F7 (SEQ ID NO 163)CTACCTACGATCTGACTAGCAAGGGGATTGGCTCCGGGTCTGGCGTGCTTGGCATCTTTGGCTTACTCTCATGTAGTTCC AMX236.C9 (SEQ ID NO 158)CTACCTACGATCTGACTAGCCAGTTCTGGGAAAAATTATTTTTTTATTTCGATCGTATTTGCTTACTCTCATGTAGTTCC AMX238.D9 (SEQ ID NO 162)CTACCTACGATCTGACTAGCCAGTTCTGGGAAAAATCATTTTTTATTTCGATCGTATTTGCTTACTCTCATGTAGTTCC AMX238.A12 (SEQ ID NO 157)CTACCTACGATCTGACTAGCCAGTTCTGGGAAAAATTATTTTTTTATTTCGATCGTATATGCTTACTCTCATGTAGTTCC

Example 2 Composition and Sequence Optimization and Sequences Example 2ATruncation of rRfY vWF Aptamers

On the basis of the vWF binding analysis described in Example 1 aboveand the cell based assay data described in Example 3 below, aptamerARC840 (AMX201.C8) (SEQ ID NO 23) was chosen from the rRfY selectionsfor further characterization.

In order to identify the core structural elements required for vWFbinding, the 3′-boundary of ARC840 (AMX201.C8) (SEQ ID NO 23) wasdetermined. The full length RNA transcript was labeled at the 5′-endwith γ-³²P ATP and T4 polynucleotide kinase. Radiolabeled ligands weresubjected to partial alkaline hydrolysis and then selectively bound insolution to human von Willebrand Factor A1 domain (SEQ ID NO 5) at 500nM before being passed through nitrocellulose filters. Both the retainedand the not retained oligonucleotides were resolved separately on 8%denaturing polyacrylamide gels. The smallest oligonucleotide bound tovWF defined the 3′-boundary. On the basis of the boundary experiments aswell as visual inspection of predicted folds, a panel of minimizedsequences was designed. Folds of all the nucleic acid sequences of theinvention were predicted using RNAstructure, Version 4.1 downloaded fromthe University of Rochester. RNAstructure is a Windows implementation ofthe Zuker algorithm for RNA secondary structure prediction based on freeenergy minimization (Mathews, D. H.; Disney, M. D.; Childs, J. L.;Schroeder, S. J.; Zuker, M.; and Turner, D. H., “Incorporating chemicalmodification constraints into a dynamic programming algorithm forprediction of RNA secondary structure,” 2004. Proceedings of theNational Academy of Sciences, US, 101, 7287-7292). RNAstructure 4.1 usesthe most current thermodynamic parameters from the Turner lab.

For the minimized rRfY aptamers, described below, the purines comprise a2′-OH and the pyrimidines comprise a 2′-F modification, while, thetemplates and primers comprise unmodified deoxyribonucleotides.

For the minimized rRf aptamer5′-GGAGCGCACUCAGCCACCCUCGCAAGCAUUUUAAGAAUGACUUGUGCCGCUGGCUG-3′ (SEQ IDNO 165), the 5′ PCR primer 5′-GATCGATCTAATACGACTCACTATA -3′ (SEQ ID NO166) and 3′ PCR primer 5′-CAGCCAGCGGCACAAGTC-3′ (SEQ ID NO 167) wereused to amplify template5′-TCGATCTAATACGACTCACTATAGGAGCGCACTCAGCCACCCTCGCAAGCATTTTAAGAATGACTTGTGCCGCTGGCTG-3′(SEQ ID NO 168).

For minimized aptamer5′-GGACCACCCUCGCAAGCAUUUUAAGAAUGACUUGUGCCGCUGGUCC-3′ (SEQ ID NO 169), 5′PCR primer 5′-GATCGATCTAATACGACTCACTATA-3′ (SEQ ID NO 166) and 3′ PCRprimer 5′-GGACCAGCGGCACAAGTC-3′ (SEQ ID NO 170) were used to amplifytemplate5′-GATCGATCTAATACGACTCACTATAGGACCACCCTCGCAAGCATTTTAAGAATGACTTGTGCCGCTGGTCC-3′(SEQ ID NO171).

For minimized aptamer5′-GGACCACCCUCGCAAGCAUUGAGAAAUGACUUGUGCCGCUGGUCC-3′ (SEQ ID NO 172), 5′PCR primer 5′-GATCGATCTAATACGACTCACTATA-3′ (SEQ ID NO 166) and 3′ PCRprimer 5′-GGACCAGCGGCACAAGTC-3′ (SEQ ID NO 170) were used to amplifytemplate5′-GATCGATCTAATACGACTCACTATAGGACCACCCTCGCAAGCATTGAGAAATGACTTGTGCCGCTGGTCC-3′(SEQ ID NO 173).

For minimized aptamer 5′-GGACCACCCUCGCAACGAGAGUUGUGCCGCUGGUCC-3′ (SEQ IDNO 174), 5′ PCR primer 5′-GATCGATCTAATACGACTCACTATA-3′ (SEQ ID NO166)and 3′ PCR primer 5′-GGACCAGCGGCACAACTC-3′ (SEQ ID NO 175) were used toamplify template5′-GATCGATCTAATACGACTCACTATAGGACCACCCTCGCAACGAGAGTTGTGCCGCTGGTCC-3′ (SEQID NO 176).

All of the above minimized aptamer sequences were transcribed,gel-purified on 15% denaturing polyacrylamide gels, 5-³²P end-labeledwith γ³²P ATP, and then desalted using two Centri-Spin 10 columns(Princeton Separations Cat.# CS-101, Adelphia, N.J.). These minimerswere primarily characterized in the cellular assays described in Example3 below.

Example 2B Truncation of rRdY vWF Aptamers

On the basis of the vWF binding analysis described in Example 1 aboveand cell based assay data described in Example 3 below, aptamersAMX203.G9 (SEQ ID NO 44) and AMX205.F7 (SEQ ID NO 49), respectively,were identified for further characterization.

In order to identify the core structural elements required for vWFbinding, the 3′-boundaries of aptamers AMX203.G9 (SEQ ID NO 44) andAMX205.F7 (SEQ ID NO 49) were determined. The full length RNAtranscripts were labeled at the 5′-end with γ-³²P ATP and T4polynucleotide kinase. Radiolabeled ligands were subjected to partialalkaline hydrolysis and then selectively bound in solution to human vWFA1 domain (SEQ ID NO 5) at 500 nM before being passed throughnitrocellulose filters. Retained oligonucleotides were resolved on 8%denaturing polyacrylamide gels. The smallest oligonucleotide bound tovWF defined the 3′-boundary. On the basis of the boundary experiments aswell as visual inspection of predicted folds using RNAstructure, Version4.1, a panel of minimized sequences was designed.

For the minimized rRdY aptamers, described below, the purines are 2′-OHpurines and the pyrimidines are deoxy-pyrimidines, while the templatesand primers comprise unmodified deoxyribonucleotides. The followingthree minimized aptamer sequences were derived from DL.159.87.70 (SEQ IDNO 44):

For minimized aptamer sequence5′-GGAGCGCACTCAGCCACGGGGTGGGTAGACGGCGGGTATGTGGCT-3′ (SEQ ID NO 177), 5′PCR primer 5′-GATCGATCTAATACGACTCACTATA-3′ (SEQ ID NO 166) and 3′ PCRprimer 5′-AGCCACATACCCGCCGTC-3′ (SEQ ID NO 178) were used to amplifyTemplate5′-GATCGATCTAATACGACTCACTATAGGAGCGCACTCAGCCACGGGGTGGGTAGACGGCGGGTATGTGGCT-3′(SEQ ID NO179).

For minimized aptamer sequence5′-GGAGCCACGGGGTGGGTAGACGGCGGGTATGTGGCTCC-3′ (SEQ ID NO 180),5′ PCRprimer 5′-GATCGATCTAATACGACTCACTATA-3′ (SEQ ID NO 166) and 3′ PCR primer5′-GGAGCCACATACCCGCCG-3′ (SEQ ID NO 181), were used to amplify template5′-GATCGATCTAATACGACTCACTATAGGAGCCACGGGGTGGGTAGACGGCGGGTATGTGGCTCC-3′(SEQ ID NO 182).

For minimized aptamer sequence

5′-GGGACGGGGTGGGTAGACGGCGGGTATGTCCC-3′ (SEQ ID NO 183),5′ PCR primer5′-GATCGATCTAATACGACTCACTATA-3′ (SEQ ID NO 166) and 3′ PCR primer5′-GGGACATACCCGCCG-3′ (SEQ ID NO 184), were used to amplify template5′-GATCGATCTAATACGACTCACTATAGGGACGGGGTGGGTAGACGGCGGGTATGTCCC-3′ (SEQ IDNO 185).

The following seven minimized aptamer sequences were derived from theaptamer according to SEQ ID NO 49:

For minimized aptamer sequence5′-GGAGCGCACTCAGCCACACGACATTGGCGGGTTGTAATTACCACGCATGGCTG-3′ (SEQ ID NO186), 5′ PCR primer 5′-GATCGATCTAATACGACTCACTATA-3′ (SEQ ID NO166) and3′ PCR primer 5′-CAGCCATGCGTGGTAATT-3′ (SEQ ID NO 187), were used toamplify template5′-GATCGATCTAATACGACTCACTATAGGAGCGCACTCAGCCACACGACATTGGCGGGTTGTAATTACCACGCATGGCTG-3′ (SEQ ID NO 188).

For minimized aptamer sequence5′-GGAGCCACACGACATTGGCGGGTTGTAATTACCACGCATGGCTCC-3′ (SEQ ID NO 189), 5′PCR primer 5′-GATCGATCTAATACGACTCACTATA-3′ (SEQ ID NO 166) and 3′ PCRprimer 5′-GGAGCCATGCGTGG-3′ (SEQ ID NO 190), were used to amplifytemplate5′-GATCGATCTAATACGACTCACTATAGGAGCCACACGACATTGGCGGGTTGTAATTACCACGCATGGCTCC-3′(SEQ ID NO 191).

For minimized aptamer sequence5′-GGAGCCACACGACATTGGCGGGCGAGAGCCACGCATGGCTCC-3′ (SEQ ID NO192), 5′ PCRprimer b 5′-GATCGATCTAATACGACTCACTATA-3′ (SEQ ID NO 166) and 3′ PCRprimer 5′-GGAGCCATGCGTGG-3′ (SEQ ID NO190), were used to amplifytemplate5′-GATCGATCTAATACGACTCACTATAGGAGCCACACGACATTGGCGGGCGAGAGCCACGCATGGCTCC-3′(SEQ ID NO 193).

For minimized aptamer sequence5′-GGAGCCACACGACATTGGCGAGAGCCACGCATGGCTCC-3′ (SEQ ID NO 194), 5′ PCRprimer 5′-GATCGATCTAATACGACTCACTATA-3′ (SEQ ID NO 166) and 3′ PCR primer5′-GGAGCCATGCGTGG-3′ (SEQ ID NO 190), were used to amplify template5′-GATCGATCTAATACGACTCACTATAGGAGCCACACGACATTGGCGAGAGCCACGCATGGCTCC-3′(SEQ ID NO 195).

For minimized aptamer sequence5′-GGAGCCACACGAGAGTGGCGGGTTGTAATTACCACGCATGGCTCC-3′ (SEQ ID NO 196), 5′PCR primer 5′-GATCGATCTAATACGACTCACTATA-3′ (SEQ ID NO 166) and 3′ PCRprimer 5′-GGAGCCATGCGTGG-3′ (SEQ ID NO 190), were used to amplifytemplate5′-GATCGATCTAATACGACTCACTATAGGAGCCACACGAGAGTGGCGGGTTGTAATTACCACGCATGGCTCC-3′(SEQ ID NO 197).

For minimized aptamer sequence5′-GGCCACACGACATTGGCGGGCGAGAGCCACGCATGGCC-3′ (SEQ ID NO 198), 5′ PCRprimer 5′-GATCGATCTAATACGACTCACTATA-3′ (SEQ ID NO 166) and 3′ PCR primer5′-GGCCATGCGTGGCTCTC-3′ (SEQ ID NO 199), were used to amplify template5′-GATCGATCTAATACGACTCACTATAGGCCACACGACATTGGCGGGCGAGAGCCACGCATGGCC-3′(SEQ ID NO 200).

For minimized aptamer sequence5′-GGAGCCACACGACATTGGCGCGAGAGCGCATGGCTCC-3′ (SEQ ID NO 201), 5′ PCRprimer 5′-GATCGATCTAATACGACTCACTATA-3′ (SEQ ID NO 166) and 3′ PCR primer5′-GGAGCCATGCGCTCTCG-3′ (SEQ ID NO 202), were used to amplify template5′-GATCGATCTAATACGACTCACTATAGGAGCCACACGACATTGGCGCGAGAGCGCATGGCTCC-3′(SEQ ID NO 203).

rRdY vWF Minimer Binding

All minimer sequences were transcribed, gel-purified on 15% denaturingpolyacrylamide gels, 5-³²P end-labeled with γ³²P ATP, and then desaltedusing two Centri-Spin 10 columns (Princeton Separations Cat.# CS-101,Adelphia, N.J.). For K_(D) determination, minimer transcripts weretested for direct binding to full length human vWF (SEQ ID NO 7), humanvWF A1 domain (SEQ ID NO 5), and rabbit vWF A1 domain (SEQ ID NO 6)using an 8 point protein titration from 0-300 nM (3 fold dilutions) in1× Dulbecco's PBS containing 0.1 mg/mL BSA (in a final volume of 50 μL)for 30 minutes at room temperature and then applied to a nitrocelluloseand nylon filter sandwich in a dot blot apparatus (Schleicher andSchuell, Keene, N.H.) K_(D) values were calculated by fitting theequation y=(max/(1+K/protein))+yint using KaleidaGraph (KaleidaGraph v.3.51, Synergy Software). Results of protein binding characterization aretabulated in Table 18.

TABLE 18 rRdY aptamer minimer binding data, only aptamers that showedpotent activity in cellular assays had their binding affinity measured.Full length Human vWF human A1 domain Rabbit vWF A1 # Minimer vWF K_(D)(nM) K_(D) (nM) domain K_(D) (nM) 1 SEQ ID NO 177 ND ND ND 2 SEQ ID NO180 1 11 14 3 SEQ ID NO 10 ± 5   ND 14 ± 7 183 4 SEQ ID NO 186 ND ND ND5 SEQ ID NO 189 1 ND ND 6 SEQ ID NO 192 2 ± 0.2 4 ± 1  8 ± 1 7 SEQ ID NO194 ND 8 SEQ ID NO 196 ND ND ND 9 SEQ ID NO 198 3 ± 0.6 5 ± 2 11 ± 2 10SEQ ID NO 201 ND ND ND (ND = not done)

Example 2C Truncation of DNA SELEX™ #1 vWF Aptamer

On the basis of the vWF binding analysis described in Example 1 above,as well as visual inspection of predicted folds, a panel of minimizedsequences was designed for the best class of binders from Family #1. Inthis case all of the binders from Family #1 are structurally related andfell into one of four mutually exclusive folds (predicted byRNAStructure 4.1) as determined by the base-pairing constraints put onthe 5′- and 3′-ends of the molecules. We synthesized and tested each ofthe four predicted folds. The sequence for each of the synthesizedminimized DNA aptamers is as follows:

SEQ ID NO 204 5′-CCAGCGGAATGAGAATGCTGATGGATTGCTCAGGTCTGCTGG-3′ ARC 845(SEQ ID NO 205) 5′ ATGAGAGTGCTGGTGGATTGCTCAGGTCTGCTGGCTGCTTACTCTCA T-3′SEQ ID NO 206 5′-CGATCTGACTAGCGGAATGAGAATGCTGGTGGATCG-3′ SEQ ID NO 2075′-GATCTGACTAGCGCAATGAGGATGCdTGATGGATTGCTCAGGTC-3′

All minimer DNA sequences were chemically synthesized, 5-³²P end-labeledwith γ-³²P ATP, and then desalted using two Centri-Spin 10 columns(Princeton Separations, Cat.# CS-101, Adelphia, N.J.). For K_(D)determination, minimers were tested for binding to human vWF A1 domain(SEQ ID NO 5) using a competition dot blot assay with a constant proteinconcentration of 10 nM and a 12 point cold competitor DNA titration (3uM, 1 uM, 333 nM, 100 nM, 33 nM, 10 nM, 3.3 nM, 1 nM, 333 pM, 100 pM,33.3 pM, 0 pM) in 1× Dulbecco's PBS containing 0.1 mg/mL BSA (finalvolume of 50 uL) for 30 minutes at room temperature. K_(D) values werecalculated by fitting the equation y=(max/(1+K/protein))+yint usingKaleidaGraph (KaleidaGraph v. 3.51, Synergy Software). Results ofprotein binding characterization are tabulated in Table 19 below. Asshown, only ARC845 of the minimized constructs retained the ability tobind to either human or rabbit vWF A1 domain

TABLE 19 DNA 1 Minimer Binding Data Full length human Rabbit vWF vWFK_(D) Human vWF A1 A1 domain # Minimer (nM) domain K_(D) (nM) K_(D) (nM)1 ARC845 = SEQ No binding 10 56 ID NO 205 2 SEQ ID NO 204 ND No bindingNo binding 3 SEQ ID NO 206 ND No binding ND 4 SEQ ID NO 207 ND Nobinding ND (ND = Not Done)

Based on these binding results, ARC 845 (SEQ ID NO 205) represents thecore nucleic acid binding sequence of the DNA SELEX™ 1, Family 1aptamers.

Example 2D DNA vWF Alternating Selection Aptamer Minimization

On the basis of the vWF binding analysis described in Example 1 aboveand cell based assay data described in Example 3 below as well as visualinspection of predicted folds for aptamers AMX237.B11 (SEQ ID NO 109)and AMX236.A12 (SEQ ID NO 127), a series of minimized sequences weredesigned. Additionally, based on the observation that aptamers AMX237.G6(SEQ ID NO 114), AMX238.E9 (SEQ ID NO 115), and AMX238.H5 (SEQ ID NO118) appeared to be slightly more potent in cellular assays, a series ofminimized sequences ARC1027-1031 (SEQ ID NOS 212-216) were synthesized.The minimized sequences according to SEQ ID NO 208 and SEQ ID NO 209represent two mutually exclusive folds predicted from the full lengthaptamer AMX237.B11 (SEQ ID NO 109).

The nucleic acid sequences for above-described minimized DNA aptamersare as follows:

(SEQ ID NO 208) 5′-GGACGATCTGACTAGCTCCAGTGTTTTATCTAATAACCGTCC-3′ (SEQ IDNO 209) 5′-GGAGCTCCAGTGTTTTATCTAATAACCGTGCGGTGCCTCCGTGAGCT CC-3′ (SEQ IDNO 210) 5′-GGAGCTGCGGTTTGGTCGACGTCAGCTCC-3′ (SEQ ID NO 211)5′-GGTAGCGACTCTGTGGAGCTGCGGTTTGG-3′ ARC1027 (SEQ ID NO 212)5′-GGCGTGCAGTGCCTATTCTAGGCCGTGCGGTGCCTCCGTCACGCC- 3T-3′ ARC1028 (SEQ IDNO 213) 5′-dGCGTGCAGTGCCT-[PEG]-AGGCCGTGCGGTGCCTCCGTCACGC C-3T-3′ARC1029 (SEQ ID NO 214)5′-GGCGTGCAGTGCC-[PEG]-GGCCGTGCGGTGCCTCCGTCACGCC- 3T-3′ ARC1030 (SEQ IDNO 215) 5′-GGCGTGCAGTGCCTATTCTAGGCCGTGCGG-[PEG]-CCGTCACGC C-3T-3′ARC1031 (SEQ ID NO 216)5′-GGCGTGCAGTGCCT-[PEG]-AGGCCGTGCGG-[PEG]-CCGTCACG CC-3T-3′

All of the above minimized aptamer sequences were chemicallysynthesized, gel-purified on 15% denaturing polyacrylamide gels and thendesalted using two Centri-Spin 10 columns (Princeton Separations Cat.#CS-101, Adelphia, N.J.) using standard methods and techniques. Theminimized sequences were characterized in cellular assays as describedin Example 3 below.

Of the initial series, SEQ ID NO 208 to SEQ ID NO 211, only SEQ ID NO208 demonstrated activity in the cellular assays (see Example 3, below).Comparison of the sequences of aptamers AMX237.B11 (SEQ ID NO 109) andAMX237.G6 (SEQ ID NO 114), AMX238.E9 (SEQ ID NO 115), and AMX238.H5 (SEQID NO 118) revealed them to be closely related and to support thepredicted secondary structure of the minimized aptamer (SEQ ID NO 208)(see FIGS. 14 and 15). These molecules, ARC1027-1031 (SEQ ID NOS212-216) further tested our hypothesis about the folding and secondarystructure of aptamers AMX237.G6 (SEQ ID NO 114), AMX238.E9 (SEQ ID NO115), and AMX238.H5 (SEQ ID NO 118) (see FIGS. 5, 14 and 15).

For K_(D) determination, the minimized sequences that showed potentactivity in the cellular assays as described in Example 3 below were5-³²P end-labeled with γ-³²P ATP, and then desalted using twoCentri-Spin 10 columns (Princeton Separations Cat.# CS-101, Adelphia,N.J.). Minimers were tested for direct binding to full length human vWF(SEQ ID NO 7), and rabbit vWF A1 domain (SEQ ID NO 6) using a 9 pointprotein titration (100 nM, 30 nM, 10 nM, 3 nM, 1 nM, 300 pM, 100 pM, 30pM, 0 pM) (see FIG. 6) in 1× Dulbecco's PBS containing 0.1 mg/mL BSA(final volume of 50 uL) for 30 minutes at room temperature and thenapplied to a nitrocellulose and nylon filter sandwich in a dot blotapparatus (Schleicher and Schuell, Keene, N.H.). K_(D) values werecalculated by fitting the equation y=(max/(1+K/protein))+yint usingKaleidaGraph (KaleidaGraph v. 3.51, Synergy Software). Results ofprotein binding characterization are tabulated in Table 20.

TABLE 20 DNA 2 aptamer minimer binding data, only aptamer minimers thatshowed potent activity in cellular assays had their binding affinitymeasured (see Example 3 below) Full length Human vWF human A1 domainRabbit vWF A1 # Minimer vWF K_(D) (nM) K_(D) (nM) domain K_(D) (nM) 1SEQ ID NO 208 ND ND ND 2 SEQ ID NO 209 ND ND ND 3 SEQ ID NO 210 ND ND ND4 SEQ ID NO 211 ND ND ND 5 ARC 1027 0.8 ND 4.6 (SEQ ID NO 212) 6 ARC10281.1 ND 3.8 (SEQ ID NO 213) 7 ARC1029 1.4 ± 0.2 ND 6.5 ± 1.5 (SEQ ID NO214) 8 ARC1030 No binding ND No binding (SEQ ID NO 215) 9 ARC1031 Nobinding ND No binding (SEQ ID NO 216) (‘ND’ = not done)

Example 2E Optimization of ARC1029 Through Aptamer Medicinal Chemistry

Highly stable and potent variants of ARC1029 (SEQ ID NO 214) wereidentified through a systematic synthetic modification approachinvolving 5 phases of aptamer synthesis, purification and assay forbinding activity. To facilitate the ease of chemical synthesis duringaptamer modification, the PEG spacer of ARC1029 (SEQ ID NO 214 wasreplaced with a short oligonucleotide sequence, dTdTdC, resulting inARC1115 (SEQ ID NO 221 )as seen in FIG. 16 and Table 21 below. A highlystabilizing 3′-inverted dT was synthesized on the three prime end ofARC1115 (SEQ ID NO 221)resulting in ARC1172 (SEQ ID NO 222) (SEQ ID NO222) also as seen in FIG. 16 and Table 21 below. Once both ARC1115 (SEQID NO 221) and ARC1172 (SEQ ID NO 222) (SEQ ID NO 222) had been shown tobind to human vWF, ARC1172 (SEQ ID NO 222) (SEQ ID NO 222) was used asthe basic template for modification as described in the Examples below.

In phase 1 of the modification process, each individual residue inARC1172 (SEQ ID NO 222) was replaced by the corresponding 2′-O methylcontaining residue (with dT being replaced by mU unless otherwisespecified) resulting in ARC 1194 (SEQ ID NO 223)-ARC1234 as shown inTable 21 below and FIG. 16. Additionally in phase 1, a set of compositereplacements were made in the stem regions of ARC1172 (SEQ ID NO 222)resulting in ARC1235 to 1243 also as shown in Table 21 and in FIG. 16.

As described herein, see e.g., in Examples 1, 2, and 3, during theprocesses of clone screening and truncation that led to ARC1029 (SEQ IDNO 214, there was excellent agreement among the relative potency ofaptamers in binding (dot-blot), FACS and BIPA assays. Accordingly,affinity for full length human vWF measured as measured in dot-blotassay binding assays was used to characterize relative affinity of themajority of the aptamer test variants synthesized.

For K_(D) determination, chemically synthesized aptamers were purifiedusing denaturing polyacrylamide gel electrophoresis, 5′end labeled withγ-³²P ATP and were tested for direct binding to full length human vWF(Calbiochem Cat.#681300, La Jolla, Calif.). An 8 point protein titrationwas used in the dot blot binding assay (100 n M, 30 nM, 10 nM, 3 nM, 1nM, 300 pm, 100 pM, 0 pM) ) in 1× Dulbecco's PBS containing 0.1 BSA(final volume of 50 uL) for 30 minutes at room temperature. K_(D) valueswere calculated by fitting the equation y=(max/(1+K/protein))+yint usingKaleidaGraph (KaleidaGraph v. 3.51, Synergy Software). Sequences of theARC1029 (SEQ ID NO 214 ) derivatives synthesized, purified and assayedfor binding to full length human vWF as well as the results of theprotein binding characterization are tabulated Table 21 below, Bindingaffinity (K_(D)) is presented in the fourth column and extent of aptamerbinding at 100 nM vWF is presented in the final column of Table 21.

TABLE 21 Phase 1 Modification Binding Results Sequence (5′ -> 3′), (NH2= 5′-hexylamine linker phosphoramidite), (3T = inv dT), (T=dT),(s=phosphorothioate), (mN = 2′-O Methyl containing % binding SEQ IDresidue), (PEG = polyethylene @ 100 nM NO: ARC # glycol), (dN=deoxyresidue) K_(D) (nM) vWF 221 ARC1115 dGdGdCdGTdGdCdAdGTdGdCd 2 36CTTdCdGdGdCdCdGTdGdCdGd GTdGdCdCTdCdCdGTdCdAdCd GdCdC 222 ARC1172dGdGdCdGTdGdCdAdGTdGdCd 2 37 CTTdCdGdGdCdCdGTdGdCdGdGTdGdCdCTdCdCdGTdCdAdCd GdCdC-3T 223 ARC1194 mGdGdCdGTdGdCdAdGTdGdCd 346 CTTdCdGdGdCdCdGTdGdCdGd GTdGdCdCTdCdCdGTdCdAdCd GdCdC-3T 224 ARC1195dGmGdCdGTdGdCdAdGTdGdCd 1 55 CTTdCdGdGdCdCdGTdGdCdGdGTdGdCdCTdCdCdGTdCdAdCd GdCdC-3T 225 ARC1196 dGdGmCdGTdGdCdAdGTdGdCd 246 CTTdCdGdGdCdCdGTdGdCdGd GTdGdCdCTdCdCdGTdCdAdCd GdCdC-3T 226 ARC1197dGdGdCmGTdGdCdAdGTdGdCd 2 46 CTTdCdGdGdCdCdGTdGdCdGdGTdGdCdCTdCdCdGTdCdAdCd GdCdC-3T 227 ARC1198 dGdGdCdGmUdGdCdAdGTdGdC 0.950 dCTTdCdGdGdCdCdGTdGdCdGd GTdGdCdCTdCdCdGTdCdAdCd GdCdC-3T 228 ARC1199dGdGdCdGTmGdCdAdGTdGdCd 19 28 CTTdCdGdGdCdCdGTdGdCdGdGTdGdCdCTdCdCdGTdCdAdCd GdCdC-3T 229 ARC1200 dGdGdCdGTdGmCdAdGTdGdCd 525 CTTdCdGdGdCdCdGTdGdCdGd GTdGdCdCTdCdCdGTdCdAdCd GdCdC-3T 230 ARC1201dGdGdCdGTdGdCmAdGTdGdCd 0.9 6 CTTdCdGdGdCdCdGTdGdCdGdGTdGdCdCTdCdCdGTdCdAdCd GdCdC-3T 231 ARC1202 dGdGdCdGTdGdCdAmGTdGdCd 0.456 CTTdCdGdGdCdCdGTdGdCdGd GTdGdCdCTdCdCdGTdCdAdCd GdCdC-3T 232 ARC1203dGdGdCdGTdGdCdAdGmUdGdC 3 40 dCTTdCdGdGdCdCdGTdGdCdGdGTdGdCdCTdCdCdGTdCdAdCd GdCdC-3T 233 ARC1204 dGdGdCdGTdGdCdAdGTmGdCd 245 CTTdCdGdGdCdCdGTdGdCdGd GTdGdCdCTdCdCdGTdCdAdCd GdCdC-3T 234 ARC1205dGdGdCdGTdGdCdAdGTdGmCd 1 50 CTTdCdGdGdCdCdGTdGdCdGdGTdGdCdCTdCdCdGTdCdAdCd GdCdC-3T 235 ARC1206 dGdGdCdGTdGdCdAdGTdGdCm 250 CTTdCdGdGdCdCdGTdGdCdGd GTdGdCdCTdCdCdGTdCdAdCd GdCdC-3T 236 ARC1207dGdGdCdGTdGdCdAdGTdGdCd 2 54 CmUTdCdGdGdCdCdGTdGdCdGdGTdGdCdCTdCdCdGTdCdAdCd GdCdC-3T 237 ARC1208 dGdGdCdGTdGdCdAdGTdGdCd 245 CTmUdCdGdGdCdCdGTdGdCdG dGTdGdCdCTdCdCdGTdCdAdCd GdCdC-3T 238 ARC1209dGdGdCdGTdGdCdAdGTdGdCd 2 44 CTTmCdGdGdCdCdGTdGdCdGdGTdGdCdCTdCdCdGTdCdAdCd GdCdC-3T 239 ARC1210 dGdGdCdGTdGdCdAdGTdGdCd 249 CTTdCmGdGdCdCdGTdGdCdGd GTdGdCdCTdCdCdGTdCdAdCd GdCdC-3T 240 ARC1211dGdGdCdGTdGdCdAdGTdGdCd 2 47 CTTdCdGmGdCdCdGTdGdCdGdGTdGdCdCTdCdCdGTdCdAdCd GdCdC-3T 241 ARC1212 dGdGdCdGTdGdCdAdGTdGdCd 249 CTTdCdGdGmCdCdGTdGdCdGd GTdGdCdCTdCdCdGTdCdAdCd GdCdC-3T 242 ARC1213dGdGdCdGTdGdCdAdGTdGdCd 6 43 CTTdCdGdGdCmCdGTdGdCdGdGTdGdCdCTdCdCdGTdCdAdCd GdCdC-3T 243 ARC1214 dGdGdCdGTdGdCdAdGTdGdCd 339 CTTdCdGdGdCdCmGTdGdCdGd GTdGdCdCTdCdCdGTdCdAdCd GdCdC-3T 244 ARC1215dGdGdCdGTdGdCdAdGTdGdCd 3 2 CTTdCdGdGdCdCdGmUdGdCdGdGTdGdCdCTdCdCdGTdCdAdCd GdCdC-3T 245 ARC1216 dGdGdCdGTdGdCdAdGTdGdCd0.6 40 CTTdCdGdGdCdCdGTmGdCdGd GTdGdCdCTdCdCdGTdCdAdCd GdCdC-3T 246ARC1217 dGdGdCdGTdGdCdAdGTdGdCd 14 18 CTTdCdGdGdCdCdGTdGmCdGdGTdGdCdCTdCdCdGTdCdAdCd GdCdC-3T 247 ARC1218 dGdGdCdGTdGdCdAdGTdGdCd 322 CTTdCdGdGdCdCdGTdGdCmGd GTdGdCdCTdCdCdGTdCdAdCd GdCdC-3T 248 ARC1219dGdGdCdGTdGdCdAdGTdGdCd 3 33 CTTdCdGdGdCdCdGTdGdCdGmGTdGdCdCTdCdCdGTdCdAdCd GdCdC-3T 249 ARC1220 dGdGdCdGTdGdCdAdGTdGdCd 1117 CTTdCdGdGdCdCdGTdGdCdGd GmUdGdCdCTdCdCdGTdCdAdC dGdCdC-3T 250 ARC1221dGdGdCdGTdGdCdAdGTdGdCd 1 43 CTTdCdGdGdCdCdGTdGdCdGdGTmGdCdCTdCdCdGTdCdAdCd GdCdC-3T 251 ARC1222 dGdGdCdGTdGdCdAdGTdGdCd 0.940 CTTdCdGdGdCdCdGTdGdCdGd GTdGmCdCTdCdCdGTdCdAdCd GdCdC-3T 252 ARC1223dGdGdCdGTdGdCdAdGTdGdCd 36 26 CTTdCdGdGdCdCdGTdGdCdGdGTdGdCmCTdCdCdGTdCdAdCd GdCdC-3T 253 ARC1224 dGdGdCdGTdGdCdAdGTdGdCd 0.547 CTTdCdGdGdCdCdGTdGdCdGd GTdGdCdCmUdCdCdGTdCdAdC dGdCdC-3T 254 ARC1225dGdGdCdGTdGdCdAdGTdGdCd 11 16 CTTdCdGdGdCdCdGTdGdCdGdGTdGdCdCTmCdCdGTdCdAdCd GdCdC-3T 255 ARC1226 dGdGdCdGTdGdCdAdGTdGdCd 1225 CTTdCdGdGdCdCdGTdGdCdGd GTdGdCdCTdCmCdGTdCdAdCd GdCdC-3T 256 ARC1227dGdGdCdGTdGdCdAdGTdGdCd 3 40 CTTdCdGdGdCdCdGTdGdCdGdGTdGdCdCTdCdCmGTdCdAdCd GdCdC-3T 257 ARC1228 dGdGdCdGTdGdCdAdGTdGdCd 243 CTTdCdGdGdCdCdGTdGdCdGd GTdGdCdCTdCdCdGmUdCdAdC dGdCdC-3T 258 ARC1229dGdGdCdGTdGdCdAdGTdGdCd 5 37 CTTdCdGdGdCdCdGTdGdCdGdGTdGdCdCTdCdCdGTmCdAdCd GdCdC-3T 259 ARC1230 dGdGdCdGTdGdCdAdGTdGdCd 346 CTTdCdGdGdCdCdGTdGdCdGd GTdGdCdCTdCdCdGTdCmAdCd GdCdC-3T 260 ARC1231dGdGdCdGTdGdCdAdGTdGdCd 1 50 CTTdCdGdGdCdCdGTdGdCdGdGTdGdCdCTdCdCdGTdCdAmCd GdCdC-3T 261 ARC1232 dGdGdCdGTdGdCdAdGTdGdCd 151 CTTdCdGdGdCdCdGTdGdCdGd GTdGdCdCTdCdCdGTdCdAdCm GdCdC-3T 262 ARC1233dGdGdCdGTdGdCdAdGTdGdCd 2 39 CTTdCdGdGdCdCdGTdGdCdGdGTdGdCdCTdCdCdGTdCdAdCd GmCdC-3T 263 ARC1234 dGdGdCdGTdGdCdAdGTdGdCd 242 CTTdCdGdGdCdCdGTdGdCdGd GTdGdCdCTdCdCdGTdCdAdCd GdCmC-3T 264 ARC1235mGmGdCmGTmGdCdAdGTdGd 13 23 CdCTTdCdGdGdCdCdGTdGdCdGdGTdGdCdCTdCdCdGTdCmAd CmGdCdC-3T 265 ARC1236 dGdGmCdGmUdGdCdAdGTdGd 332 CdCTTdCdGdGdCdCdGTdGdCd GdGTdGdCdCTdCdCdGTmCdA mCdGmCmC-3T 266ARC1237 mGmGmCmGmUmGdCdAdGTd 41 9 GdCdCTTdCdGdGdCdCdGTdGdCdGdGTdGdCdCTdCdCdGTmCm AmCmGmCmC-3T 267 ARC1238 dGdGdCdGTdGdCdAdGTmGdCd2 43 CTTdCmGmGdCdCdGTdGdCdG dGTdGdCdCTdCdCdGTdCdAdCd GdCdC-3T 268ARC1239 dGdGdCdGTdGdCdAdGTdGmC 5 37 mCTTdCdGdGmCdCdGTdGdCdGdGTdGdCdCTdCdCdGTdCdAd CdGdCdC-3T 269 ARC1240 dGdGdCdGTdGdCdAdGTmGmC 440 mCTTdCmGmGmCdCdGTdGdCd GdGTdGdCdCTdCdCdGTdCdAd CdGdCdC-3T 270 ARC1241dGdGdCdGTdGdCdAdGTdGdCd no no CTTdCdGdGdCdCdGTdGdCmG binding bindingmGTdGdCdCTdCdCmGTdCdAdC dGdCdC-3T 271 ARC1242 dGdGdCdGTdGdCdAdGTdGdCd nono CTTdCdGdGdCdCdGTdGmCdGd binding binding GTdGdCdCTmCmCdGTdCdAdCdGdCdC-3T 272 ARC1243 dGdGdCdGTdGdCdAdGTdGdCd no noCTTdCdGdGdCdCdGTdGmCmG binding binding mGTdGdCdCTmCmCmGTdCdAd CdGdCdC-3T

As can be seen from the binding data in Table 21, the positions thatmost readily tolerate substitution of a deoxy residue for a 2′-O methylresidue correlate well with the sequence conservation mapped onto thesecondary structure of ARC1029 (SEQ ID NO 214 shown in FIG. 15 thusproviding further, independent support for the proposed structure of theaptamer. The positions of ARC1172 (SEQ ID NO 222) that do not tolerate2′-O-Me modifications as well as the positions that do are shown in FIG.15B.

Based upon the structure activity relationship (SAR) results of theindividual and composite deoxy to methoxy aptamers described immediatelyabove in the phase 1 modification process, a second series of aptamerswas designed, synthesized, purified and tested for binding to vWF. Forthese and all subsequent aptamers, molecules that retained an affinity(K_(D)) of ˜10 nM or better as well as an extent of binding at 100 nMvWF of at least 35% were the goal. ARC1338 (SEQ ID NO 273)-ARC1348 (SEQID NO 283), as shown in FIG. 17 and Table 21, were synthesized duringphase 2 of the modification process. ARC1338 (SEQ ID NO 273) to 1342were synthesized with block modifications based on the toleratedindividual substitutions from phase 1 modification. ARC1343 (SEQ ID NO278)-ARC1345 were synthesized each with a different phosphorothioatephosphate backbone modification (see FIG. 17 and Table 22 below) Lastly,ARC1346 (SEQ ID NO 281)-ARC1348 (SEQ ID NO 283) were synthesized to testremoving a single base pair from stem 1, stem 2 and from both stems ofARC1342 as shown in FIG. 17 and Table 22 below.

TABLE 22 Phase 2 Modification Binding Results Sequence (5′ -> 3′), (NH2= 5′-hexylamine linker phosphoramidite), (3T = inv dT), (T=dT),(s=phosphorothioate), (mN = 2′-O Methyl containing SEQ ID residue), (PEG= polyethylene % binding @ NO: ARC # glycol), (dN=deoxy residue) K_(D)(nM) 100 nM vWF 273 ARC1338 mGmGmCmGmUdGdCdAdGTdGd 7.4 21CdCTTdCdGdGdCdCdGTdGdCdG dGTdGdCdCTdCdCdGTdCmAmC mGmCmC-3T 274 ARC1339dGdGdCdGTdGdCdAmGmUdGdC 2.4 39 dCTTdCdGdGdCdCmGTmGdCdGdGTdGdCdCTdCdCmGmUmCdAdC dGdCdC-3T 275 ARC1340 dGdGdCdGTdGdCdAdGTdGdCdC7.4 43 TTdCdGdGdCdCdGTmGdCdGdGT mGmCdCmUdCdCmGmUmCdAdC dGdCdC-3T 276ARC1341 mGmGmCmGmUdGdCdAmGmUm 22.5 26 GmCmCmUmUmCmGmGmCdCdGTdGdCdGdGTdGdCdCTdCdCdG TdCmAmCmGmCmC-3T 277 ARC1342mGmGmCmGmUdGdCdAmGmUm 15.6 33 GmCmCmUmUmCmGmGmCdCmGTmGdCdGdGTmGmCdCmUdCd CmGmUmCmAmCmGmCmC-3T 278 ARC1343 mGmGmCmGmUdG-s-23.9 21 dCdAmGmUmGmCmCmUmUmC mGmGmC-s-dCmGTmGdCdG-s- dGTmGmCdCmUdC-s-dCmGmUmCmAmCmGmCmC-3T 279 ARC1344 mGmGmCmGmUdG-s-dC-s- 4.8 17dAmGmUmGmCmCmUmUmCmG mGmC-s-dCmGTmGdC-s-dG-s- dGTmGmCdCmUdC-sdCmGmUmCmAmCmGmCmC-3T 280 ARC1345 mGmGmCmGmU-s-dG-s-dC-s- 12.1 29dAmGmUmGmCmCmUmUmCmG mGmC-s-dCmG-s-TmG-s-dC-s-dG-s-dG-s-TmGmC-s-dCmU-s-dC-s dCmGmUmCmAmCmGmCmC-3T 281 ARC1346mGmCmGmUdGdCdAmGmUmGm 11 51 CmCmUmUmCmGmGmCdCmGT mGdCdGdGTmGmCdCmUdCdCmGmUmCmAmCmGmC-3T 282 ARC1347 mGmGmCmGmUdGdCdAmGmUm no noGmCmUmUmCmGmCdCmGTmGd binding binding CdGdGTmGmCdCmUdCdCmGmUmCmAmCmGmCmC-3T 283 ARC1348 mGmCmGmUdGdCdAmGmUmGm no noCmUmUmCmGmCdCmGTmGdCd binding binding GdGTmGmCdCmUdCdCmGmUm CmAmCmGmC-3T

As seen in Table 22, the results from phase 2 of aptamer modificationrevealed ARC1346 (SEQ ID NO 281) to be the most potent of the highlysubstituted ARC1029 (SEQ ID NO 214 derivative aptamers generated thusfar. Interestingly as shown by the results with ARC1347 (SEQ ID NO 282)and ARC1348 (SEQ ID NO 283), removal of a base pair from stem 2 is nottolerated in this highly modified context.

ARC1361 (SEQ ID NO 284) to ARC1381 (SEQ ID NO 304), shown in Table 23and FIG. 17, were synthesized during phase 3 of the aptamer modificationprocess. As the dG to mG substitution at position 6 was poorly toleratedin test variants in phase 1 aptamer modification and guanosine atposition 6 pairs with the cytidine at position 36, ARC1346 (SEQ ID NO281) was synthesized with a mC to dC modification at position 36resulting in ARC1361 (SEQ ID NO 284) as shown in Table 23 below. ARC1361(SEQ ID NO 284) served as the base sequence for introduction of singlephosphorothioate phosphate backbone modifications that resulted inARC1362 to ARC1381 (SEQ ID NO 304) also shown in Table 23 below.

TABLE 23 Phase 3 Modification Binding Results Sequence (5′ -> 3′), (NH2= 5′-hexylamine linker phosphoramidite), (3T = inv dT), (T=dT),(s=phosphorothioate), (naN = 2′-O Methyl containing SEQ ID residue),(PEG = polyethylene % binding @ NO: ARC # glycol), (dN=deoxy residue)K_(D) (nM) 100 nM vWF 222 ARC1172 dGdGdCdGTdGdCdAdGTdGdCdC 2 37TTdCdGdGdCdCdGTdGdCdGdGT dGdCdCTdCdCdGTdCdAdCdGdCd C-3T 284 ARC1361mGmCmGmUdGdCdAmGmUmGm 7.9 38.5 CmCmUmUmCmGmGmCdCmGTmGdCdGdGTmGmCdCmUdCdCm GmUdCmAmCmGmC-3T 285 ARC1362 mGmCmGmU-s- 9.9 34.3dGdCdAmGmUmGmCmCmUmUm CmGmGmCdCmGTmGdCdGdGT mGmCdCmUdCdCmGmUdCrnAmCmGmC-3T 286 ARC1363 mGmCmGmUdG-s- 12.7 32.7 dCdAmGmUmGmCmCmUmUmCmGmGmCdCmGTmGdCdGdGTm GmCdCmUdCdCmGmUdCmAmC mGmC-3T 287 ARC1364mGmCmGmUdGdC-s- 8.2 36.9 dAmGmUmGmCmCmUmUmCmG mGmCdCmGTmGdCdGdGTmGmCdCmUdCdCmGmUdCmAmCmG mC-3T 288 ARC1365 mGmCmGmUdGdCdA-s- 10.8 35.4mGmUmGmCmCmUmUmCmGmG mCdCmGTmGdCdGdGTmGmCdC mUdCdCmGmUdCmAmCmGmC- 3T 289ARC1366 mGmCmGmUdGdCdAmGmUmGm 15.5 28.9 CmCmUmUmCmGmGmC-s-dCmGTmGdCdGdGTmGmCdCmU dCdCmGmUdCmAmCmGmC-3T 290 ARC1367mGmCmGmUdGdCdAmGmUmGm 13.9 30.4 CmCmUmUmCmGmGmCdC-s-mGTmGdCdGdGTmGmCdCmUdC dCmGmUdCmAmCmGmC-3T 291 ARC1368mGmCmGmUdGdCdAmGmUmGm 1.8 38.2 CmCmUmUmCmGmGmCdCmG-s-TmGdCdGdGTmGmCdCmUdCdC mGmUdCmAmCmGmC-3T 292 ARC1369mGmCmGmUdGdCdAmGmUmGm 16.3 26.2 CmCmUmUmCmGmGmCdCmGTmGdCdGdGTmGmCdCmUdCdCm GmUdCmAmCmGmC-3T 293 ARC1370mGmCmGmUdGdCdAmGmUmGm 10.1 22.5 CmCmUmUmCmGmGmCdCmGT mG-s-dCdGdGTmGmCdCmUdCdCmGm UdCmAmCmGmC-3T 294 ARC1371 mGmCmGmUdGdCdAmGmUmGm8.4 32.1 CmCmUmUmCmGmGmCdCmGT mGdC-s- dGdGTmGmCdCmUdCdCmGmUdCmAmCmGmC-3T 295 ARC1372 mGmCmGmUdGdCdAmGmUmGm 23.5 35.2CmCmUmUmCmGmGmCdCmGT mGdCdG-s- dGTmGmCdCmUdCdCmGmUdCm AmCmGmC-3T 296ARC1373 mGmCmGmUdGdCdAmGmUmGm 7.1 33.0 CmCmUmUmCmGmGmCdCmGT mGdCdGdG-s-TmGmCdCmUdCdCmGmUdCmA mCmGmC-3T 297 ARC1374 mGmCmGmUdGdCdAmGmUmGm 9.527.2 CmCmUmUmCmGmGmCdCmGT mGdCdGdGT-s- mGmCdCmUdCdCmGmUdCmAm CmGmC-3T298 ARC1375 mGmCmGmUdGdCdAmGmUmGm 8.8 25.5 CmCmUmUmCmGmGmCdCmGTmGdCdGdGTmGmC-s- dCmUdCdCmGmUdCmAmCmGm C-3T 299 ARC1376mGmCmGmUdGdCdAmGmUmGm 4.4 31.3 CmCmUmUmCmGmGmCdCmGT mGdCdGdGTmGmCdC-s-mUdCdCmGmUdCmAmCmGmC- 3T 300 ARC1377 mGmCmGmUdGdCdAmGmUmGm 7.4 30.9CmCmUmUmCmGmGmCdCmGT mGdCdGdGTmGmCdCmU-s dCdCmGmUdCmAmCmGmC-3T 301ARC1378 mGmCmGmUdGdCdAmGmUmGm 9.1 31.1 CmCmUmUmCmGmGmCdCmGTmGdCdGdGTmGmCdCmUdC-s dCmGmUdCmAmCmGmC-3T 302 ARC1379mGmCmGmUdGdCdAmGmUmGm 10.4 31.3 CmCmUmUmCmGmGmCdCmGTmGdCdGdGTmGmCdCmUdCdC-s mGmUdCmAmCmGmC-3T 303 ARC1380mGmCmGmUdGdCdAmGmUmGm 12.0 32.5 CmCmUmUmCmGmGmCdCmGTmGdCdGdGTmGmCdCmUdCdCm GmU-s-dCmAmCmGmC-3T 304 ARC1381mGmCmGmUdGdCdAmGmUmGm 8.7 35.8 CmCmUmUmCmGmGmCdCmGTmGdCdGdGTmGmCdCmUdCdCm GmUdC-s-mAmCmGmC-3T

As shown in Table 23 above, while the majority of the modificationstested in phase 3 had little or no beneficial effect, ARC1368 (SEQ ID NO291), which contains a single phosphorothioate modification betweenmG-20 and dT-21 binds to human vWF with an affinity identical (withinexperimental error) to that of the parent compound, ARC1172 (SEQ ID NO222).

During phase 4 and phase 5 aptamer modification, ARC1524 (SEQ ID NO 305)to ARC1535 (SEQ ID NO 316), ARC1546 (SEQ ID NO 317) and ARC1759 (SEQ IDNO 318), shown in FIG. 18, were synthesized. A circular permutation ofthe sequence that closed stem 1 and opened stem 2 as illustrated in FIG.19 was synthesized.

As shown in Table 24 below, many of these aptamers bound to vWF,however, none were as potent as ARC1368 (SEQ ID NO 291). Interestingly,though consistent with the SAR generated in Phase 1 of aptamermodification, ARC1525, containing only a single change from dT to mT atposition 27, showed no binding at all to vWF. ARC1525 was used as anegative control in many of the biological assays in which ARC1368 (SEQID NO 291) was subsequently tested. Again, consistent with the SAR datafrom Phase 3 of aptamer modification, ARC1759 (SEQ ID NO 318) which isidentical to ARC1172 (SEQ ID NO 222) except that it has singlephosphorothioate substitution between the G at position 21 and the T atposition 22 showed measurable improvement in affinity relative toARC1172 (SEQ ID NO 222).

TABLE 24 Phase 4 and 5 Aptamer Modification Binding Results Sequence (5′-> 3′), (NH2 = 5′-hexylamine linker phosphoramidite), (3T = inv dT),(T=dT), (s=phosphorothioate), (mN = 2′-O Methyl containing SEQ IDresidue), (PEG = polyethylene K_(D) % binding @ NO: ARC # glycol), (dN =deoxy residue) (nM) 100 nM vWF 222 ARC1172 dGdGdCdGTdGdCdAdGTdGdCdC 2 37TTdCdGdGdCdCdGTdGdCdGdGT dGdCdCTdCdCdGTdCdAdCdGdCd C-3T 291 ARC1368mGmCmGmUdGdCdAmGmUmGm 1.8 38.2 CmCmUmUmCmGmGmCdCmG-s-TmGdCdGdGTmGmCdCmUdCdC mGmUdCmAmCmGmC-3T 305 ARC1524mGmCmGmUdGdCdAmGmUmGm 5.7 26.5 CmCmUmUmCmGmGmCdCmG-s-TmGdCdGdGTmGmCdCmUdCdC mGmUmCmAmCmGmC-3T 306 ARC1525mGmCmGmUdGdCdAmGmUmGm No No CmCmUmUmCmGmGmCdCmGm binding bindingTmGdCdGdGTmGmCdCmUdCdC mGmUmCmAmCmGmC-3T 307 ARC1526mGmCmGmUdGdCdAmGmUmGm 4.7 29.0 CmCmUmUmUmGmGmCdCmG-s-TmGdCdGdGTmGmCdCmUdCdC mGmUmCmAmCmGmC-3T 308 ARC1527mGmCmGmUdGdCdAmGmUmGm 3.4 14.1 CmCPEGmGmGmCdCmG-s-TmGdCdGdGTmGmCdCmUdCdC mGmUdCmAmCmGmC-3T 309 ARC1528mGmCmGmUdGdCdAmGmUmGm 4.2 10.2 CmCPEGmGmGmCdCmG-s-TmGdCdGdGTmGmCdCmUdCdC mGmUmCmAmCmGmC-3T 310 ARC1529mCmGmUdGdCdAmGmUmGmCm 12.3 22.6 CmUmUmCmGmGmCdCmG-s-TmGdCdGdGTmGmCdCmUdCdC mGmUmCmAmCmG-3T 311 ARC1530 mCmGmUdGdCdAmGmUmGmCm15.0 19.2 CmUmUmUmGmGmCdCmG-s- TmGdCdGdGTmGmCdCmUdCdC mGmUmCmAmCmG-3T312 ARC1531 mCmGmUdGdCdAmGmUmGmCm 1.5 20.0 CPEGmGmGmCdCmG-s-TmGdCdGdGTmGmCdCmUdCdC mGmUdCmAmCmG-3T 313 ARC1532 mCmGmUdGdCdAmGmUmGmCm2.6 19.3 CPEGmGmGmCdCmG-s- TmGdCdGdGTmGmCdCmUdCdC mGmUmCmAmCmG-3T 314ARC1533 mCmGmGmCdCmG-s- 4.0 31.7 TmGdCdGdGTmGmCdCmUdCdCmGmUmCmAmCPEGmGmUdGdC dAmGmUmGmCmCmG-3T 315 ARC1534 mCmGmGmCdCmG-s- 62.825.6 TmGdCdGdGTmGmCdCmUdCdC mGmUmCmAmCmUmUmUmGm UdGdCdAmGmUmGmCmCmG-3T316 ARC1535 mCmCmGmGmCdCmG-s 27.1 48.3 TmGdCdGdGTmGmCdCmUdCdCmGmUmCmAmCmUmUmUmGm UdGdCdAmGmUmGmCmCmGmG- 3T 317 ARC1546mCmCmGmGmCdCmG-s- 24 26 TmGdCdGdGTmGmCdCmUdCdC mGmUdCmAmCmGmUmUmCmCmGmUdGdCdAmGmUmGmCmCm GmG-3T 318 ARC1759 dGdGdCdGTdGdCdAdGTdGdCdC 0.7 46TTdCdGdGdCdCdG-s- TdGdCdGdGTdGdCdCTdCdCdGT dCdAdCdGdCdC-3T

Example 2F Conjugation of PEG Moieties to Modified Aptamers

Polyethylene glycol moieties were conjugated to the 5′ terminus ofARC1368 (SEQ ID NO 291) and ARC1172 (SEQ ID NO 222) via amine reactivechemistries. The oligonucleotides ARC1635NH2-mGmCmGmUdGdCdAmGmUmGmCmCmUmUmCmGmGmCdCmG-s-dTmGdCdGdGdTmGmCdCmUdCdCmGmUdCmAmCmGmC-3T(ARC1368 (SEQ ID NO 291) with a 5′ hexylamine modification) and ARC1884(SEQ ID NO 322)NH2-dGdGdCdGdTdGdCdAdGdTdGdCdCTdTdCdGdGdCdCdGTdGdCdGdGTdGdCdCTdCdCdGTdCdAdCdGdCdC-3T(ARC1172 (SEQ ID NO 222) with a 5′ hexylamine modification) werechemically synthesized.

The amine-modified aptamers were conjugated to different PEG moieties,as indicated in Table 25 below, post-synthetically.

TABLE 25 Hexylamine modified or PEG conjugated aptamers Sequence (5′ ->3′), (NH2 = 5′-hexylamine linker phosphoramidite), (3T = inv dT),(T=dT), (s=phosphorothioate), (mN = 2′-O Methyl containing SEQ IDresidue), (PEG = polyethylene NO: ARC # glycol), (dN=deoxy residue) 319ARC1635 NH2- mGmCmGmUdGdCdAmGmUmGm CmCmUmUmCmGmGmCdCmG-s-TmGdCdGdGTmGmCdCmUdCdC mGmUdCmAmCmGmC-3T 320 ARC1779 PEG20K-NH2-mGmCmGmUdGdCdAmGmUmGm CmCmUmUmCmGmGmCdCmG-s- TmGdCdGdGTmGmCdCmUdCdCmGmUdCmAmCmGmC-3T 321 ARC1780 PEG40K-NH2- mGmCmGmUdGdCdAmGmUmGmCmCmUmUmCmGmGmCdCmG-s- TmGdCdGdGTmGmCdCmUdCdC mGmUdCmAmCmGmC-3T 322ARC1884 NH2- dGdGdCdGTdGdCdAdGTdGdCdC TTdCdGdGdCdCdGTdGdCdGdGTdGdCdCTdCdCdGTdCdAdCdGdCd C-3T 323 ARC1885 PEG20K-NH2-dGdGdCdGTdGdCdAdGTdGdCdC TTdCdGdGdCdCdGTdGdCdGdGTdGdCdCTdCdCdGTdCdAdCdGdCd C-3T

Example 3 Functional Cell Assays

Biological vWF Dependent Assays

The effectiveness of various aptamers in blocking vWF function inseveral biological assays is described in this Example.

In one assay botrocetin is used. Botrocetin, a protein isolated fromsnake venom, is known to induce von Willebrand Factor binding to thegpIb receptor on live and fixed platelets. This reaction causesagglutination of suspensions of fixed platelets via vWF multimerization.In preparations of platelet rich plasma (hereinafter “PRP”),vWF/botrocetin induction of agglutination is followed by a second phaseof platelet aggregation caused by metabolic activation of the platelets.These two reactions: vWF binding to fixed platelets and vWF mediatedplatelet aggregation, can be used to measure the activity of aptamers ofthe invention.

The amount of vWF bound to fixed platelets can be measured with anantibody to vWF. The fluorescence signal from bound antibody incubatedwith a fluorescein conjugated secondary antibody is then detected andquantified by flow cytometry. The ability of an aptamer of the inventionto block vWF binding to platelets is correlated with a reduction influorescence signal.

Botrocetin induces the binding of the A1 domain of vWF to platelets, aswell as the full length protein. It was determined by the inventors that6-Histidine-tagged rabbit A1 domain vWF purified protein could beinduced to bind to human lyophilized platelets with botrocetin. RabbitA1 binding to platelets is measured with an anti-poly-His antibodyfollowed by incubation with a phycoerythrin conjugated secondaryantibody. The degree of binding can be quantified by flow cytometricanalysis. The ability of aptamers to block the binding of rabbit A1 tohuman fixed platelets was correlated with decreased fluorescence signal.

In platelet rich plasma isolated from fresh human blood, botrocetininduces platelet aggregation via vWF. Platelet aggregate formation canbe measured optically as an increase in % light transmittance on aChronolog Model 490-4D Aggregometer because aggregation of plateletsclarifies the plasma. Aptamers of the invention were analyzed for theirability to inhibit botrocetin induced platelet aggregation (“BIPA”) inhuman blood. An aptamer of the invention was considered to be active ifit could prevent aggregate formation for six minutes post botrocetinaddition.

Another assay of this Example, using the PFA-100 instrument, is anagonist independent but vWF dependent assay that uses the PFA-100instrument (Harrison et al., Clin. Lab. Haem., v 24, p 225-32 (2002)).The PFA-100 simulates the formation of a hemostatic plug underconditions of high shear force in vivo by recording the time requiredfor a platelets to aggregate and block the flow of citrated whole bloodthrough a microscopic aperture in a membrane coated with collagen andeither epinephrine or ADP. This activity is von Willebrand factordependent as high MW vWF multimers bind to immobilized collagen on themembrane and then bind to and activate platelets because of the shearforce induced by drawing the blood through the microscopic aperture.Thus this assay is complimentary to the BIPA and FACS assays in that itis vWF dependent, however it has some advantages in that it does notrequire the addition of the vWF agonist botrocetin and uses whole bloodinstead of platelet rich plasma.

Another assay of this Example used ADP to induce platelet aggregation.Aggregation of platelet rich plasma (PRP) can be in induced in multipleways. The snake venom protein botrocetin acts on vWF as described above,stabilizing its interaction with the platelet receptor gpIb therebyinducing platelet aggregation. Binding of vWF to gpIb is an early stepin platelet aggregation, thus there is an expectation that inhibitorsthat block downstream components of the aggregation process (i.e., theIIbIIIa antagonists Integrelin™ and ReoPro™) would also preventbotrocetin induced platelet aggregation. However, in the case ofagonists that act directly on platelets and induce aggregation (ADP forexample), one would expect that antagonists upstream of the agonistwould be ineffective (an anti-vWF aptamer for example), whileantagonists that act directly on platelets (IIbIIIa antagonists) wouldremain potent. The specificity of a vWF antagonist relative to a IIbIIIaantagonist will increase the safety of the anti-vWF antagonist bydecreasing the bleeding time associated with treatment. For patientswith atherosclerotic plaques in stenosed arteries, platelet aggregationoccurs as platelets bind to collagen immobilized vWF on the surface ofthe plaque. Thus both inhibiting the vWF/gpIb interaction as well asblocking the IIbIIIa receptor binding to fibrin will prevent plateletaggregation. The biological specificity conferred by targeting vWFinsures that unlike anti-IIbIIIa treatment, platelets themselves are nottargeted directly insuring they can still be activated by other means,thus reducing potential bleeding complications associated withanti-platelet therapy.

The following materials were used in Examples 3A-3D described below:human von Willebrand Factor (vWF) (SEQ ID NO 7), and bovine serumalbumin were purchased from Calbiochem (Cat#681300 and #126593,respectively) (La Jolla, Calif.); domain A1 rabbit vWF (SEQ ID NO 6) wasexpressed and purified using standard methods and conditions.Lyophilized human platelets (P/N 299-2), cuvettes (P/N 312), stir bars(P/N 311), platelet aggregometer (model 490-4D), and AGGRO/LINK Softwarewere purchased from Chronolog (Haverton, Pa.). Botrocetin (12201-100U-B)was manufactured by Pentapharm (Basel, Switzerland). Fresh blood wasobtained from apparently healthy, nonsteroidal anti-inflammatory drug(“NSAID”) free donors and was drawn into 5 mL 0.105 M Sodium CitrateVacutainer tubes (Cat#369714) (Becton Dickinson-Franklin Lakes, N.J.).Physiological saline was manufactured by Aldon (Cat #9420306) (Avon,N.Y.) and phosphate buffered saline (Cat#21-040-CV) was purchased fromCellgro (Herndon, Va.). Flow cytometric experiments were performed on aBD Biosciences FACSCAN machine and analyzed with CellQuest Software (SanJose, Calif.). Anti-von Willebrand Factor mouse monoclonal antibody(Cat# GTI-V1A) was purchased from GTI (Waukesha, Wis.). Penta-HIS-biotinconjugate monoclonal antibody (Cat#34440) was purchased from Qiagen(Germany). Anti-mouse IgG2a -FITC conjugate (Cat#553390) was purchasedfrom BD Biosciences (San Diego, Calif.). Anti-mouse IgG-PE conjugateantibody (Cat#715-116-150) was purchased from Jackson ImmunoResearchLaboratories (West Grove, Pa.).

Example 3A Full Length Human von Willebrand Factor Platelet BindingAssay

Aptamer potency to block human vWF binding to lyophilized platelets wasassessed by flow cytometric analysis. Titrations of aptamers (0 nM, 0.1nM to 1000 nM) were pre-incubated briefly with 5 nM of full length humanvWF in FACS buffer (PBS plus 0.5% bovine serum albumin) at roomtemperature in a volume of 50 uL. Another 50 uL containing 5 uL oflyophilized platelets plus 1 μL of 0.1 U/μL of botrocetin in FACS bufferwas added to aptamer/vWF. This reaction was allowed to proceed for 15minutes at 37 degrees C. after which 200 uL of FACS buffer was added.Platelets were collected by a 6 minute spin at 1470 RCF and thesupernatant was discarded. The pellets were resuspended in 100 uL ofFACS buffer containing a 1:100 dilution of anti-vWF antibody and wereincubated at room temperature for 30 minutes. After dilution with 200 μLof FACS buffer, the platelets were spun at 1470 RCF for 6 minutes andthe supernatant was discarded. The pellets were resuspended in a 1:100solution of anti-IgG2a-FITC antibody and were incubated in the dark for30 minutes at room temperature. The entire 100 uL was diluted into 200uL of FACS buffer and analyzed immediately by flow cytometric analysisin the FACSCAN. Artifactual data from contaminating debris waseliminated from the analysis by drawing a gate around the population ofsingle and aggregated platelets. Mean fluorescent intensity (“MFI”)readings were quantified for each sample analyzed by flow cytometry.Background MFI was subtracted from all data points. Percent inhibitionwas reported by calculating the percent value of binding of full lengthhuman vWF to platelets in the presence of aptamer at a givenconcentration relative to binding in the absence of any aptamer (seeFIG. 7). IC₅₀ values were determined by fitting the percent inhibitionof vWF binding to platelets as a function of aptamer concentration tothe equation:% inhibition=% inhibition_(max)/(1+IC ₅₀/aptamer conc.)

Results of botrocetin induced vWF binding characterization are tabulatedin Table 26 below.

Example 3B Rabbit von Willebrand Factor Domain A1 Platelet Binding Assay

The ability of aptamers of the invention to block rabbit vWF domain A1binding to lyophilized platelets was also assessed by flow cytometricanalysis. Titrations of aptamers (zero, 0.1 nM to 1000 nM) werepreincubated briefly with 4 nM of rabbit A1 vWF in FACS buffer (PBS plus0.5% bovine serum albumin) at room temperature in a volume of 50 uL.Another 50 uL containing 5 uL of lyophilized platelets plus 1 uL of 0.1U/uL of botrocetin in FACS buffer was added to aptamer/vWF. Thisreaction was allowed to proceed for 15 minutes at 37 degrees C. afterwhich 200 uL of FACS buffer was added. Platelets were collected by a 6minute spin at 1470 RCF and the supernatant was discarded. The pelletswere resuspended in 100 uL of FACS buffer containing a 1:200 dilution ofanti-Penta-HIS-biotin conjugate antibody and were incubated at roomtemperature for 30 minutes. After the dilution with 200 uL of FACSbuffer, the platelets were spun at 1470 RCF for 6 minutes, and thesupernatant was discarded. The pellets were resuspended in a 1:100solution of anti-IgG-PE antibody and were incubated in the dark for 30minutes at room temperature. The entire 100 uL was diluted into 200 uLof FACS buffer and analyzed immediately by flow cytometric analysis inthe FACSCAN. Contaminating debris was eliminated from the analysis bydrawing a gate around the population of single and aggregated plateletsand collecting data from 10000 events. Median fluorescent intensity(“MedFI”) readings (which are generally equivalent to the MFI readings,described in Example 3a above, for comparative purposes) were quantifiedfor each sample analyzed by flow cytometry. Background MedFI wassubtracted from all data points. Percent inhibition was reported bycalculating the percent value of binding of full length human vWF toplatelets in the presence of aptamer at a given concentration relativeto binding in the absence of any aptamer (see FIG. 7). IC₅₀ values weredetermined by fitting the percent inhibition of vWF binding to plateletsas a function of aptamer concentration to the equation:% inhibition=% inhibition_(max)/(1+IC ₅₀/aptamer conc.)Results of botrocetin induced rabbit A1 vWF binding characterization aretabulated below in Table 26.

TABLE 26 Results of FACS and BIPA Assays (‘ND’ = not done) Inhibition of≦200 nM Inhibition of full length rabbit VWF A1 aptamer gives hVWFbinding in FACS domain binding >90% assay in FACS assay blocking inAverage IC₅₀ Average BIPA at 6 Aptamer ID IC₅₀ range IC₅₀ range IC₅₀minutes rRdY aptamer aptamers (AMX203.D6) no activity ND ND SEQ ID NO 31(AMX205.H8) no activity ND ND SEQ ID NO 32 (AMX205.H11) no activity NDND SEQ ID NO 33 (AMX205.D11) no activity ND ND SEQ ID NO 35 (AMX206.F9)no activity ND ND SEQ ID NO 36 (AMX206.H9) no activity ND ND SEQ ID NO37 (AMX206.A10) no activity ND ND SEQ ID NO 38 (AMX205.F9) no activityND ND SEQ ID NO 39 (AMX206.E7) no activity ND ND SEQ ID NO 40(AMX206.D7) no activity ND ND SEQ ID NO 41 (AMX203.A1) no activity ND NDSEQ ID NO 43 (AMX203.G9) 1.2 nM to 8.5 nM 4.7 nM 152 pM to 592 pM yesSEQ ID NO 44 = ARC 842 1.6 nM (AMX205.H9) SEQ ID NO no activity ND ND 45(AMX206.D8) SEQ ID NO no activity no activity ND 46 (AMX205.F7) SEQ IDNO 321 pM to 1.6 nM 818 pM 1.5 nM to 4.7 nM yes 49 = ARC 841 7.1 nM(AMX205.H10) SEQ ID NO no activity no activity ND 50 rRdY aptamerminimers SEQ ID NO 177 1.6 nM to 5.4 nM 3 nM ND ND SEQ ID NO 180 731 pMto 3.8 nM 2 nM 8.2 nM to 9.3 nM yes 10.5 nM SEQ ID NO 183 6.6 nM to 99nM 26.5 nM ND ND SEQ ID NO 186 971 pM to 2.2 nM 1.3 nM ND ND SEQ ID NO189 564 pM to 1.6 nM 1.1 nM ND ND SEQ ID NO 192 1.1 nM to 5.3 nM 2.6 nM2 nM to 2.2 nM yes 2.3 nM SEQ ID NO 194 no activity ND ND SEQ ID NO 196no activity ND ND SEQ ID NO 198 14 nM to 25 nM 20.6 nM 8.5 nM to 12.6 nMND 16.8 nM SEQ ID NO 201 1.3 nM to 370 nM 150 nM ND no activity rRfYaptamer aptamers (AMX201.B1) no activity ND yes SEQ ID NO 11 (AMX198.G1)no activity ND ND SEQ ID NO 12 (AMX201.H3) no activity ND yes SEQ ID NO13 (AMX201.G1) no activity ND ND SEQ ID NO 15 (AMX201.C8) 24 pM to 1.8nM 528 pM 141 pM to 682 pM yes SEQ ID NO 23 = ARC 840 704 pM rRfYaptamer minimers SEQ ID NO 165 562 pM to 14.4 nM 6.4 nM ND ND SEQ ID NO169 103 pM to 17 nM 6.9 nM ND ND SEQ ID NO 172 1.8 nM to 7.4 nM 4.3 nMND ND SEQ ID NO 174 1.3 nM to 12.7 nM 6.6 nM 4.9 nM to 6.8 nM yes 5 nMDNA SELEX 1, minimer ARC845 no activity no activity no activity noactivity no activity SEQ ID NO 205 DNA SELEX 2 aptamer aptamersAMX237.E10 no activity ND ND SEQ ID NO 138 AMX237.G7 2.3 nM to 14 nM 6.5nM 12.3 nM ND SEQ ID NO 134 AMX.236.G1 8.6 nM to 72 nM 46 nM ND SEQ IDNO 164 AMX237.A11 466 pM to 5.6 nM 2.2 nM 4.7 nM ND SEQ ID NO 98AMX237.A2 815 pM to 7.3 nM 3.5 nM 7 nM ND SEQ ID NO 99 AMX238.D12 684 pMto 3.4 nM 2 nM 7.2 nM ND SEQ ID NO 100 AMX238.G5 273 pM to 2.6 nM 1.1 nM5.5 nM ND SEQ ID NO 106 AMX238.E9 772 pM to 6.5 nM 3.4 nM ND ND SEQ IDNO 115 AMX238.H5 514 pM to 860 pM 658 pM ND ND SEQ ID NO 118 AMX237.G61.5 nM to 2.5 nM 1.9 nM ND yes SEQ ID NO 114 AMX237.B11 151 pM to 4.8 nM2.5 nM 5.7 nM ND SEQ ID NO 109 AMX236.A12 1.2 nM to 17 nM 10.7 nM 14.5nM ND SEQ ID NO 127 DNA SELEX 2 aptamer minimers SEQ ID NO 208 1.6 nM to10.2 nM 4.3 nM ND ND SEQ ID NO 209 no activity ND ND SEQ ID NO 210 noactivity ND ND SEQ ID NO 211 no activity ND ND ARC1027 208 pM to 4.2 nM1.5 nM 462 pM to 2 nM yes SEQ ID NO 212 4.2 nM ARC1028 473 pM to 2.7 nM1.2 nM 526 pM to 1.5 nM yes SEQ ID NO 213 2.7 nM ARC1029 333 pM to 1.1nM 609 pM 490 pM to 754 pM yes SEQ ID NO 214 979 pM ARC1030 no activityND ND SEQ ID NO 215 ARC1031 no activity ND ND SEQ ID NO 216

Example 3C Inhibition of Botrocetin Induced Platelet Aggregation (BIPAAssay)

In order to determine the activity of aptamers on live human platelets,BIPA assays were done using freshly prepared platelet rich plasma. Bloodwas obtained from healthy human donors who had not taken NSAIDS for atleast five days. 21¾ gauge butterfly needles (Cat#367287) from BectonDickinson were used to draw blood into 0.105 M sodium citrate vacutainertubes. Collected blood was pooled into 15 mL conical tubes and was spunat 200 g for 20 minutes. The turbid, yellow layer of platelet richplasma (“PRP”) was withdrawn from the tubes, pooled, and set aside atroom temperature. The remaining blood was spun at 2500 g for tenminutes. The clarified layer of plasma, known as platelet poor plasma(“PPP”), was withdrawn and set aside at room temperature. PRP wasaliquoted into cuvettes containing stir bars at a volume of 470 uL. Asample of 500 uL of PPP was aliquoted into a cuvette and placed in thePPP reference cell of the platelet aggregometer. Samples of PRP wereprewarmed at 37 degrees C. in the platelet aggregometer for 3-5 minutesbefore being used in BIPA assays. First, the concentration of botrocetinneeded to induce platelet aggregation for each individual donor wasdetermined by titration. This concentration of botrocetin was used forthe remainder of the experiment. Next, aptamer was assayed by addingtitrations of aptamer (zero, 1 nM to 1000 nM) to prewarmed PRP for oneminute, followed by addition of botrocetin. With platelet aggregation,an increase in amplitude of light transmission is seen (FIG. 8). Theconcentration at which platelet aggregation is blocked at 90% or greaterafter 6 minutes is reported in the final column of Table 26. Using theAggro/LINK Software, area under the curve (“AUC”) can be generated fromthe aggregometer trace and used to calculate percent inhibition ofaptamer at any given concentration on botrocetin induced plateletaggregation as seen in FIG. 9 for Aptamer ARC1029 (SEQ ID NO 214).

Example 3D Biological Activity of Selected Modified Aptamers in a Seriesof Biological Assays

ARC1172 (SEQ ID NO 222), ARC1346 (SEQ ID NO 281), ARC1368 (SEQ ID NO291), ARC1525 (negative control), ARC1779 (SEQ ID NO 320), ARC1780 (SEQID NO 321), and ARC1885 (SEQ ID NO 323) identified in the medchemmodification process described in Example 2 above, were tested in aseries of biological assays. These assays included the FACS (asdescribed in Example 3A and 3B) and BIPA assays (as described in Example3C), as well as the platelet PFA-100 assay described below.

Materials:

The following materials were used in the platelet function analyzer(PFA) assay: Fresh whole blood was collected from healthy non-steroidalanti-inflammatory drug (NSAID) free donors into 5 ml 0.105M sodiumcitrate tubes (Cat#369714, Becton Dickenson) using 21¾ gauge butterflyneedles (Cat#367287, Becton Dickenson). Fresh whole blood was collectedfrom healthy non-steroidal anti-inflammatory drug (NSAID) freecynomolgus macaques. Aptamers were diluted with physiological salinefrom Aldon (Cat#9420306) in no-additive vacutainer tubes (Cat#366434,Becton Dickenson). Samples were loaded onto collagen/epinephrine testcartridges (Cat# B4170-20A, Dade Behring) which were used in the PFA-100machine (Dade Behring). Trigger solution (Cat# B4170-50, Dade Behring)was used in the self-test and to pre-wet the test cartridges. O-ringcleaning pads (Cat# B4170-73, Dade Behring) were used in the self-testand in the O-ring cleaning process.

PFA Assay:

A self-test, which included an O-ring cleaning process, was always runon the PFA-100 machine before running any samples to ensure properfunction of the machine.

Fresh whole blood was collected from healthy donors or cynomolgusmacaques, as indicated in Table 27 below, who/that had not taken NSAIDsfor at least three days. Blood from human donors was collected into 5 ml0.105M sodium citrate tubes using a 21 ¾ gauge butterfly needle, and thetubes were gently inverted three times to ensure mixture of blood withsodium citrate. During the entire experiment, the tubes of whole bloodwere gently inverted every five minutes to prevent settling.

In order to assay the titration of aptamer in whole blood, the aptamerwas added to no-additive vacutainer tubes and diluted to the desiredconcentrations (ex: 0 nM, 1 nM to 1000 nM) using physiological salinesuch that the final volume was 60 μl. When the PFA-100 was ready to runthe next set of samples, 1940 μl of whole blood was added to the tubecontaining a concentration of aptamer. This tube was gently invertedthree times to thoroughly mix the aptamer and blood. Samples were alwaysrun in duplicate on the PFA-100 machine. 800 μl of this blood mixturewas loaded into the collagen/epinephrine test cartridges. The testcartridges were loaded onto the PFA-100 machine. The time of occlusionof the aperture was measured by the PFA-100, with a maximal time of 300seconds. We estimate the IC₉₅ in this assay to be the minimumconcentration of aptamer that extends the closing time to 300 seconds.

FIG. 20 depicts clotting time in human whole blood as a function ofaptamer concentration in the PFA-100 assay for ARC1368 (SEQ ID NO 291)and the negative control ARC1525. Additional results of the FACS, BIPAand PFA-100 assays are tabulated in Table 27 below.

TABLE 27 FACS, BIPA and PFA-100 results FACS IC₅₀ ~IC₉₀ ~IC₉₅ ~IC₉₅ vsFACS BIPA PFA-100 PFA-100 human IC₅₀ vs (nM) with (nM) with (nM) withfull rabbit human citrated citrated length A1 platelet human C. macaquevWF domain rich whole whole ARC # (nM) (nM) plasma blood blood ARC1172 22 ~200 ~100 ND (SEQ ID NO 222) ARC1346 50 180 >1000 ND ND ARC1368 2 4.0~200 ~100 ~100 ARC1525 ND ND no no ND inhibition inhibition ARC1779 NDND ~100 ~100 ND ARC1780 ND ND ~100 ~100 ND ARC1885 ND ND ~50 ~100 ND

As expected, there was a strong correlation among the observedaffinities of aptamers for vWF in the binding assay described in Example2 above and their relative potency in the biological assays. Thenon-binding negative control ARC1525 did not display activity in anyassay in which it was tested

Example 3E ARC1368, Integrilin™ and ReoPro™ in BIPA, PFA-100 and AIPAAssays

The potency of ARC1368 (SEQ ID NO 291), Integrillin™ and ReoPro™ wereevaluated in human whole blood in PFA-100, in human PRP in BIPA (asdescribed above in Examples 3D and 3C respectively) and ADP InducedPlatelet Aggregation (AIPA) assays. AIPA was performed with human PRPexactly as was done for BIPA described above in Example 3C with theexception that instead of adding botrocetin, 10 micromolar ADP(Chronolog, Haverton, Pa.) was added to induce platelet aggregation. ThePFA-100 results are shown in FIG. 21. The BIPA results are shown in FIG.22. The AIPA results are shown in FIG. 23. As can be seen in FIGS. 21and 22, ARC1368 (SEQ ID NO 291) shows potency comparable to ReoPro™ inPFA-100 and BIPA assays. Consistent with the vWF dependent mechanismdescribed above, the anti-vWF aptamer shows no ability to block AIPAwhile the IIbIIIa antagonists remain potent in that assay as shown inFIG. 23.

Example 4 Pharmacokinetic Studies

In Examples 4 and 5, all mass based concentration data refers only tothe molecular weight of the oligonucleotide portion of the aptamer,irrespective of the mass conferred by PEG conjugation.

Example 4A Stability of Anti-vWF Aptamers in Human and Rat Plasma

ARC1172 (SEQ ID NO 222), ARC1346 (SEQ ID NO 281), ARC1368 (SEQ ID NO291) and ARC1533 were assayed for nuclease stability in both human andrat plasma. Plasma nuclease degradation was measured on denaturingpolyacrylamide gel electrophoresis as described below. Briefly, forplasma stability determination, chemically synthesized aptamers werepurified using denaturing polyacrylamide gel electrophoresis, 5′endlabeled with γ-³²P ATP and then gel purified again. Trace 32-P labeledaptamer was incubated in the presence of 100 nM unlabeled aptamer in 95%human or rat plasma in a 200 microliter binding reaction. The reactionfor the time zero point was made separately with the same componentsexcept that the plasma was replaced with PBS. This insured that theamount or radioactivity loaded on gels was consistent across anexperiment. Reactions were incubated at 37° C. in a thermocycler for the1, 3, 10, 30 and 100 hours unless otherwise specified. At each timepoint, 20 microliters of the reaction was removed, combined with 200microliters of formamide loading dye and flash frozen in liquid nitrogenand stored at −20° C. After the last time point was taken, frozensamples were thawed and 20 microliters was removed from each time point.SDS was then added to the small samples to a final concentration of0.1%. The samples were then incubated at 90° C. for 10-15 minutes andloaded directly onto a 15% denaturing PAGE gel and run at 12 W for 35minutes. Radioactivity on the gels was quantified using a Storm 860phosphoroimager system. The percentage of full length aptamer at eachtime point was determined by quantifying the full length aptamer bandand dividing by the total counts in the lane. The fraction of fulllength aptamer at each time-point was then normalized to the percentagefull length aptamer of the 0 hour time-point. The fraction of fulllength aptamer as a function of time was fit to the equation:m1*e^(−m2*m0)

-   -   where m1 is the maximum % full length aptamer (m1=100); and m2        is the rate of degradation. The half-life of the aptamer        (T_(1/2)) is equal to the (1n 2)/m2.

Sample data for human plasma is shown in FIG. 24 and the results for theaptamers tested are summarized in Table 28. Consistent with ourexpectations, aptamers are more stable in human plasma than in ratplasma and increasing the number of 2′-OMe modifications correlates withincreasing plasma stability.

TABLE 28 Aptamer Plasma Stability half-life T½ T½ Human Rat plasmaplasma ARC # (hrs) (hrs) ARC1172 17  3 (SEQ ID NO 222) ARC1346 not 19done ARC1368 63 21 ARC1533 93 not done

Example 4B PK/PD of PEGylated Derivatives of ARC1368 in CynomolgusMacaques

ARC1368 (SEQ ID NO 291), 1779 (SEQ ID NO 320) and 1780 (SEQ ID NO 321)(as described in Example 2 above) were injected intravenously intocynomolgus macaques (n=3/group) at a dosage of 3 mg/kg which wasexpected to yield an instantaneous plasma concentration of 3 uM,approximately 30-fold higher than the putative effective dose.Subsequently, citrated blood samples were collected at regular intervalsand processed for plasma.

To demonstrate that the aptamers were pharmacologically active in vivo,Botracetin-induced platelet aggregation (BIPA) was performed 5 minutespost-dosing, at the presumed plasma C_(max). All animals had completeinhibition of BIPA at this point demonstrating that the aptamers werefunctional in vivo.

Subsequently, plasma aptamer concentrations were determined using theOligreen assay (Gray et al., Antisense and Nucleic Acid Drug Development7 (3):133-140 (1997). The data were subsequently analyzed using theprogram WinNonlin to yield the pharmacokinetic parameters listed inTables 29 to 31 below.

Additionally, the primate plasma aptamer concentration plotted as afunction of time is depicted in the graph of FIG. 25. The meanconcentration-times profiles based on the OliGreen™ assay showed thatthe pharmacokinetic profiles of ARC1368 (SEQ ID NO 291), ARC1780 (SEQ IDNO 321) and ARC1779 (SEQ ID NO 320) were mainly monophasic. TheunPEGylated aptamer (ARC1368 (SEQ ID NO 291)) displayed a rapiddistribution phase compared to ARC1779 (SEQ ID NO 320) and ARC1780 (SEQID NO 321). Unlike ARC1368 (SEQ ID NO 291), the 40 kDa PEG conjugate ARC1780 (SEQ ID NO 321) displayed prolonged distribution phase compared toARC1779 (SEQ ID NO 320). ARC1779 (20 kDa PEG) displayed a distributionphase with α-half-life of ˜2 hr.

TABLE 29 NonCompartmental Pharmacokinetic Parameter Estimates forARC1368 After 3 mg/kg IV Administration in Monkeys Based on OligreenAssay Data PK Parameter Unit 1101 1102 1103 Mean StdDev Tmax hr 0.080.08 0.08 0.08 0.00 Cmax ng/mL 26409 19170 23301 22960 3632 AUC0-lasthr*ng/mL 23096 31926 20650 25224 5932 MRTlast hr 4.50 5.46 3.48 4.480.99

TABLE 30 NonCompartmental Pharmacokinetic Parameter Estimates forARC1779 After 3 mg/kg IV Administration in Monkeys Based on OligreenAssay Data PK Parameter Unit 2101 2103 2104 Mean StdDev Tmax hr 0.250.50 0.25 0.33 0.14 Cmax ng/mL 69065 65717 70344 68375 2389 AUClasthr*ng/mL 309245 336590 235503 293779 52288 MRTlast hr 5.27 4.66 2.694.21 1.35

TABLE 31 NonCompartmental Pharmacokinetic Parameter Estimates forARC1780 After 3 mg/kg IV Administration in Monkeys Based on OligreenAssay Data PK Parameter Unit 3101 3102 3103 Mean StdDev Tmax hr 2.002.00 0.25 1.42 1.01 Cmax ng/mL 55965 37320 50690 47992 9611 AUClasthr*ng/mL 740559 613180 899455 751065 143426 AUCall hr*ng/mL 740559613180 899455 751065 143426 MRTlast hr 8.69 9.54 14.11 10.78 2.91

Following the Oligreen assay analysis, plasma aptamer concentrationswere determined for the animals dosed with ARC1779 (SEQ ID NO 320) usinga validated HPLC-based assay. The HPLC data were analyzed vianoncompartmental and 2-compartment analysis using the program WinNonlin.Reanalysis of the monkey samples with a more sensitive HPLC methodgenerated a concentration-times profile of ARC1779 (SEQ ID NO 320) to bebiphasic showing both distribution and elimination phase Consistent withthe results observed using the OliGreen assay, the HPLC-based resultsindicated the distribution half-life (t_(1/2α)) was 1.4 h and aelimination half-life was 12.9 (t_(1/2β)) for ARC1779 (SEQ ID NO 320).

Example 4C PK/PD of ARC1779 in Cynomolgus Macaques

-PK/PD correlation of ARC1779 was evaluated in three cynomolgus macaquesafter a single intravenous (IV) bolus dose at 0.5 mg/kg. ARC1779 levelsin the plasma were correlated to PD effects of ARC1779 in inhibition ofplatelet function or prolongation of cutaneous bleeding time (CBT).

Following IV administration, blood was collected percutaneously atvarious time points post-dose for PK and PD analysis. PD effect ofARC1779 on platelet function was determined by PFA-100 assays and effectof ARC1779 on bleeding was measured by CBT time. In addition, plasmasamples were analyzed by HPLC for quantitation of ARC1779 levels. PKparameter estimates were determined by 2-compartment analysis. Theresults are presented in FIG. 26.

The concentration-time profiles generated for individual monkeys showedpredominantly the distribution phase of ARC1779 pharmacokinetics Thedistribution half-life was determined to be approximately 1.0 hour(t_(1/2α)). The elimination half-life (t_(1/2β)), was notwell-determined from the available data.

The PD effect of ARC1779 on platelet aggregation measured by the PFA-100assay is shown in FIG. 26. When the plasma concentrations of ARC1779were in excess of 300 nM, platelet function was inhibited as assessed bythe PFA-100 instrument. However, when plasma aptamer concentrationsdecreased to approximately 77 nM, platelet function returned to normal.

As also seen in FIG. 26, the PD effect of ARC1779 on bleeding timeprolongation was found to be minimal in these studies.

In summary, ARC1779 inhibited platelet function in vivo at a plasmaconcentration of approximately 300 nM. This in vivo concentration wasapproximately 3-fold higher than the observed concentration of aptamernecessary to inhibit platelet function in vitro. In contrast, even athigh plasma concentrations (1000 nM), ARC1779 showed minimal effect oncutaneous bleeding time following single bolus dosing.

Example 5 Functional Animal Assays

In Examples 4 and 5 described herein, all mass based concentration datarefers only to the molecular weight of the oligonucleotide portion ofthe aptamer, irrespective of the mass conferred by PEG conjugation.

Example 5A Pharmacodynamics of ARC1779 in Cynomolgus Macaques

C. macaques were dosed at 0.5 mg/kg IV bolus with ARC1779 (SEQ ID NO320). PFA-100 closure time, BIPA and cutaneous bleeding time (“CBT”)were all measured as a function of time throughout the studies. BIPA andPFA-100 closure times were measured as described in previously inExample 3. Cutaneous bleeding times were measured using standardprotocols described as follows.

A blood pressure cuff was applied to the biceps region of the forearm tobe tested and inflated to maintain a constant pressure of 40 mmHg. Usinga Surgicutt® Automated Incision Device (ITC, Edison, N.J.), alongitudinal incision was made over the lateral aspect of the volarsurface of the forearm distal to the antecubital crease. A stopwatch wasstarted at the time of incision. At 15-30 seconds post incision, theblood was wicked with Surgicutt® Bleeding Time Blotting Paper (ITC,Edison, N.J.) while avoiding direct contact with the incision. Every15-30 seconds the blotting paper was rotated and re-blotted at a freshsite on the paper. The blotting was repeated until blood was no longerwicked onto the paper or for 30 minutes whichever came first. Bleedingtime is determined to within 30 seconds of the time when blood is nolonger wicked onto the paper.

The table of FIG. 27 shows the cutaneous bleeding time in minutes, BIPAIC₉₀ in nM and PFA IC₉₅ in nM at various time points, shown in column 1,relative to ARC1779 (SEQ ID NO 320) dosing for three different animals.FIG. 28 shows a graph of the average PFA-100 closure time from the bloodof three ARC1779 (SEQ ID NO 320) treated macaques taken at various timepoints following dosing. FIG. 29 shows the cutaneous bleeding time ofthe three ARC1779 (SEQ ID NO 320) treated macaques taken at various timepoints following dosing. FIG. 30 correlates the average cutaneousbleeding time in ARC1779 (SEQ ID NO 320) treated C. macaques (leftvertical axis) to the PFA-100 closure time (right vertical axis). Asshown FIGS. 27 to 30, at time points up to and including 2 hours, whereBIPA and PFA-100 closure time were maximally inhibited, there is verylittle increase in cutaneous bleeding times. At concentrations of theanti-gpIIbIIIa antagonist Integrilin™ that yield similar inhibition ofplatelet aggregation ex vivo, template or cutaneous bleeding times arebetween 20 and 30 minutes. See, e.g. Phillips, D. R. and Scarborough, R.M., Am J Cardiol, 80(4A):11B-20B (1997). While not wishing to be boundby any particular theory, these data are consistent with and supportiveof our hypothesis that an anti-vWF A1 domain aptamer antagonist willblock platelet activity in vivo by blocking platelets from binding tovWF immobilized at sites of vascular damage without inhibiting plateletfunction and thus without increasing cutaneous bleeding times.

Example 5B Assessment of ARC1779 in a Cynomolgus Macaque ElectrolyticThrombosis Model

A study was performed to test the efficacy of ARC1779 (SEQ ID NO 320) ininhibiting intra-arterial thrombosis in a well documented non-humanprimate electrolytic thrombosis model. See, e.g., Rote et al., Stroke,1994: 25, 1223-1233. Thirteen cynomolgus monkeys were divided into fourgroups and assigned to a treatment regimen as indicated in table 30below.

TABLE 32 Electrolytic Thrombosis Study Design Group Number of Dose DoseNecropsy Number Animals Test Article (mg/kg) Dose Regime Volume Day 1 3Vehicle DVE to Group 3 IV bolus, ~15 minutes prior to ≦10 mL Day 0(saline) initiation of electrical injury followed by continuous infusionon Day 0 2 1 ReoPro ™ 0.25 mg/kg IV bolus, once on Day 0, ~15 (chimeric7E3) minutes prior to initiation of electrical injury 3 5 ARC1779 0.61mg/kg IV bolus,, ~15 minutes prior to bolus + 0.0037 mg/kg/mininitiation of electrical injury, infusion followed by continuousinfusion on Day 0 4 4 ReoPro ™ 0.25 mg/kg IV bolus,, ~15 minutes priorto (chimeric 7E3) bolus + 0.125 μg/kg/min initiation of electricalinjury, infusion followed by continuous infusion on Day 0 DVE = DoseVolume Equivalent, IV = Intravenous

Each animal was anesthetized prior to surgical preparation, intubatedand maintained in anesthesia with isoflurane inhalant anesthetic toeffect delivered through a volume-regulated respirator. An intravenouscatheter was also placed in a peripheral vein for administration oflactated Ringer's solution during the procedure.

A catheter was placed in the femoral artery of each animal forcontinuous monitoring of arterial blood pressure. Similarly a catheterwas placed in the femoral vein for blood sample collection. Each carotidartery was instrumented with a Doppler flow probe connected to a flowmeter. The flow probes were placed around the artery at a point proximalto the insertion of the intra-arterial electrode and stenosis. Thestenosis was placed around each carotid artery so that the blood flowwas reduced by approximately 50% to 60% without altering the mean bloodflow. Blood flow in the carotid arteries was monitored and recordedcontinuously throughout the observation periods.

Electrolytic injury to the intimal surface of each carotid artery wasaccomplished via placement of an intravascular electrode. Each electrodewas connected to the positive pole of a constant current device andcathode connected to a distant subcutaneous site. Continuous current wasdelivered to each vessel for a period of 3 hours or for 30 minutes aftercomplete occlusion, whichever was shorter.

Once the electrodes were placed on the right carotid artery (“RCA”) theanimal was administered the test article as indicated in Table 32 above.Approximately 15 minutes after test article administration, theelectrical current was applied at 100 μA. Blood samples and CBTmeasurements were collected at the time points specified in Blood SampleCollection Schedule as indicated in FIG. 31. The current was terminated˜30 minutes after the blood flow signal remained stable at zero flow(which indicates an occlusive thrombus had been formed at the site) orafter 180 minutes of electrical stimulation. Approximately 195 minutesafter the test article was administered, the left carotid artery (“LCA”)had electrical current administered in a similar fashion as previouslydescribed for the RCA. After termination of all surgical procedures andsample collection each animal was euthanized.

The ARC1779 (SEQ ID NO 320) plasma concentration (as measured by HPLC)over time for each animal in treatment group 3 is depicted in FIG. 32.The time to occlusion measured via Doppler flow for each treatment groupis indicated in FIG. 33. As can be determined from FIG. 33, ARC1779 (SEQID NO 320) inhibited thrombus formation during sequential 180-minuteelectrical injuries to the carotid arteries of the cynomolgus monkeys inthis animal thrombosis model.

Example 5C Assessment of ARC1779 at Various Doses in a CynomolgusMacaque Electrolytic Thrombosis Model

The study described in Example 5B above was extended to test theefficacy of ARC1779 (SEQ ID NO 320) in inhibiting intra-arterialthrombosis in the non-human primate electrolytic thrombosis model atlower aptamer dosage levels.

An additional 10 cynomolgus monkeys (2.5 to 3.5 kg) were divided intotreatment groups 5 to 7 and were treated according the regimen indicatedin Table 33 below.

TABLE 33 Study Design Group No. of Dose Necropsy Number Animals TestArticle Dosage Level Volume Dosing Regimen Day 1 3 Vehicle DVE to ≦10 mLIV bolus, ~15 minutes prior to Day 0 (Saline) Group 3 initiation ofelectrical injury, followed by continuous infusion on Day 0 2 1Abciximab 0.25 mg/kg IV bolus, once on Day 0 ~15 minutes (ReoPro) priorto initiation of electrical injury 4 5 Abciximab 0.25 mg/kg bolus IVbolus, ~15 minutes prior to (ReoPro) and 0.125 μg/kg/min initiation ofelectrical injury, followed by continuous infusion on Day 0 3 5 ARC17790.61 mg/kg bolus ≦10 mL IV bolus, ~15 minutes prior to Day 0 andinitiation of electrical injury, followed 0.0037 mg/kg/min by continuousinfusion on Day 0 infusion (1000 nM) 5 2 ARC1779 0.123 mg/kg bolus ≦10mL IV bolus, ~15 minutes prior to Day 0 and initiation of electricalinjury, followed 0.001 mg/kg/min by continuous infusion on Day 0infusion (300 nM) 6 4 ARC1779 0.2 mg/kg bolus and ≦10 mL IV bolus, ~15minutes prior to Day 0 0.00165 mg/kg/min initiation of electricalinjury, followed infusion (500 nM) by continuous infusion on Day 0 7 4ARC1779 0.298 mg/kg bolus ≦10 mL IV bolus, ~15 minutes prior to Day 0and initiation of electrical injury, followed 0.00248 mg/kg/min bycontinuous infusion on Day 0 infusion (750 nM)

The animal procedure for each treatment group in Table 33 was conductedas reported for the animals in Example 5B. The time to occlusionmeasured via Doppler flow for each treatment group is indicated in FIG.33.

Without any platelet antagonist therapy (control animals) 100% ofcarotid arteries develop occlusive thrombi within 60 minutes. Incontrast, only 20% or arteries developed occlusive thrombi in animalstreated with Reopro. In the ARC1779 treatment groups at infusion ratestargeted to reach constant plasma levels of 1000 nM, 750 nM, 500 nM and300 nM, 0%, 25%, 63% and 100% of carotid arteries developed occlusivethrombi. In addition to occlusive thrombus formation, we also assessedthe effect of ARC1779 on cutaneous bleeding times which is shown in FIG.34. As can be seen in FIG. 34, at some doses and/or time points in thisanimal model prolonged CBT was observed, while at other doses and timepoints CBT was not prolonged.

The invention having now been described by way of written descriptionand example, those of skill in the art will recognize that the inventioncan be practiced in a variety of embodiments and that the descriptionand examples above are for purposes of illustration and not limitationof the following claims.

1. An aptamer that binds to a von Willebrand Factor target, wherein theaptamer modulates von Willebrand Factor-mediated platelet adhesion,activation and/or aggregation and the aptamer isPEG20K-NH2-mGmCmGmUdGdCdAmGmUmGmCmCmUmUmCmGmGmCdCmG-s-TmGdCdGdGTmGmCdCmUdCdCmGmUdCmAmCmGmC-3T(SEQ ID No. 291) (ARC1779), where “NH2” is a 5′-hexylamine linkerphosphoramidite, “3T” is an inverted deoxythymidine, “T” is a dT, “s” isa phosphorothioate backbone modification, “mN” is a 2′-O Methylcontaining residue, “PEG” is a polyethylene glycol and “dN” is a deoxyresidue, and wherein the aptamer comprises a K_(D) for von WillebrandFactor of less than 100 nM.
 2. The aptamer of claim 1, wherein the vonWillebrand Factor target is a human von Willebrand Factor target.
 3. Theaptamer of claim 1, wherein the aptamer inhibits a function of the vonWillebrand Factor target in vitro.
 4. An aptamer that binds to a vonWillebrand Factor target, wherein the aptamer nucleic acid sequence isPEG20K-NH2-mGmCmGmUdGdCdAmGmUmGmCmCmUmUmCmGmGmCdCmG-s-TmGdCdGdGTmGmCdCmUdCdCmGmUdCmAmCmGmC-3T(SEQ ID No. 291) (ARC 1779), where “NH2” is a 5′-hexylamine linkerphosphoramidite, “3T” is an inverted deoxythymidine, “T” is a dT, “s” isa phosphorothioate backbone modification, “mN” is a 2′-O Methylcontaining residue, “PEG” is a polyethylene glycol and “dN” is a deoxyresidue.
 5. An aptamer that binds to a von Willebrand Factor target,wherein the aptamer modulates von Willebrand Factor-mediated plateletadhesion, activation and/or aggregation and the aptamer isPEG20K-NH2-mGmCmGmUdGdCdAmGmUmGmCmCmUmUmCmGmGmCdCmG-s-TmGdCdGdGTmGmCdCmUdCdCmGmUdCmAmCmGmC-3T(SEQ ID No 291) (ARC 1779), where “NH2” is a 5′-hexylamine linkerphosphoramidite, “3T” is an inverted deoxythymidine, “T” is a dT, “s” isa phosphorothioate backbone modification, “mN” is a 2′-O Methylcontaining residue, “PEG” is a polyethylene glycol and “dN” is a deoxyresidue.
 6. An aptamer having the structure set forth below:

wherein: n is about 454 ethylene oxide units (PEG=20 kDa)

 is a linker, and the aptamer ismGmCmGmUdGdCdAmGmUmGmCmCmUmUmCmGmGmCdCmG-s-TmGdCdGdGTmGmCdCmUdCdCmGmUdCmAmCmGmC-3T(SEQ ID NO: 291) wherein “mN” is a 2′-OMe containing residue, “T” is adT, “dN” is a deoxy nucleotide, the “s” is a phosphorothioate backbonemodification and “3T” is an inverted deoxy thymidine.
 7. The aptamer ofclaim 6, wherein the linker is an alkyl linker.
 8. The aptamer of claim7, wherein the alkyl linker comprises 2 to 18 consecutive CH₂ groups. 9.The aptamer of claim 7, wherein the alkyl linker comprises 2 to 12consecutive CH₂ groups.
 10. The aptamer of claim 7, wherein the alkyllinker comprises 3 to 6 consecutive CH₂ groups.
 11. The aptamer of claim10, having the structure set forth below:

wherein: n is about 454 ethylene oxide units (PEG=20 kDa), and theaptamer ismGmCmGmUdGdCdAmGmUmGmCmCmUmUmCmGmGmCdCmG-s-TmGdCdGdGTmGmCdCmUdCdCmGmUdCmAmCmGmC-3T(SEQ ID NO: 291) wherein “mN” is a 2′-OMe containing residue, “T” is adT, “dN” is a deoxy nucleotide, the “s” is a phosphorothioate backbonemodification and “3T” is an inverted deoxy thymidine.
 12. Apharmaceutical composition comprising a therapeutically effective amountof the aptamer of claim 1 or a salt thereof, and a pharmaceuticallyacceptable carrier or diluent.
 13. An aptamer comprisingPEG20K-NH2-mGmCmGmUdGdCdAmGmUmGmCmCmUmUmCmGmGmCdCmG-s-TmGdCdGdGTmGmCdCmUdCdCmGmUdCmAmCmGmC-3T(SEQ ID No. 291) (ARC 1779), wherein “NH2” is a 5′-hexylamine linkerphosphoramidite, “3T” is an inverted deoxythymidine, “T” is a dT, “s” isa phosphorothioate backbone modification, “mN” is a 2′-O Methylcontaining residue, “PEG” is a polyethylene glycol and “dN” is a deoxyresidue.
 14. The aptamer of claim 4, wherein the von Willebrand Factortarget is a human von Willebrand Factor target.
 15. A pharmaceuticalcomposition comprising a therapeutically effective amount of the aptamerof claim 4 or a salt thereof, and a pharmaceutically acceptable carrieror diluent.
 16. The aptamer of claim 5, wherein the von WillebrandFactor target is a human von Willebrand Factor target.
 17. Apharmaceutical composition comprising a therapeutically effective amountof the aptamer of claim 5 or a salt thereof, and a pharmaceuticallyacceptable carrier or diluent.
 18. A pharmaceutical compositioncomprising a therapeutically effective amount of the aptamer of claim6or a salt thereof, and a pharmaceutically acceptable carrier ordiluent.
 19. A pharmaceutical composition comprising a therapeuticallyeffective amount of the aptamer of claim 13 or a salt thereof, and apharmaceutically acceptable carrier or diluent.